116 Draft Triclo

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GREEN BOOK 1

Triclosan

CIR EXPERT PANEL MEETING

AUGUST 30-31, 2010



To: CIR Expert Panel Me

From: Director, CIR

Subject: Triclosan Draft Repo

Date 30 July 2010

The literature review for Triclosan wasbook. They have been addressed in pre

In addition, 3 new sources of informati

on Consumer Products opinion on tricl

(same committee, new name) opinion

safety by Rodricks, et al. published in

consideration. The Rodricks, et al. pub

of numbered citations and author and d

incarnation, but for purposes of this rev

The approach CIR has taken to evaluat

the need to focus on the putative issuesface of EPA, FDA, Europe, and Austra

or an efficient use of time. Each of the

Rodricks et al. review and the SCCS an

The issues that were identified in the sc

dioxin impurities; photostability and di

bacterial resistance. In addition to thes

risk assessment: establishing a NOAE

there additional issues that should have

If the above 6 issues (and any newly idthat the CIR Expert Panel could issue a

identified and CIR would issue a forma

Memorandum

mbers and Liaisons

rt

issued in April. Several comments were received and arparing the draft report for your review.

n have been incorporated: the European Commission Sc

san, the European Commission Scientific Committee on

n triclosan antimicrobial susceptibility, and a critical revi

ritical Reviews in Toxicology. The European opinions a

lication will be provided to you as a .pdf. One note: the r

ate citations. We will make them all numbered citations

iew, we figured you could handle a mix.

the safety of triclosan in cosmetics differs from the nor

and in part because adding one more summary of relevaia already having done such reviews) was not considere

previously available reviews discussed most of the same

d SCCP opinions just added to the glut of reviews.

ientific literature review were: triclosan exposure; triclo

xin photoproducts; carcinogenicity; endocrine disruptio

6 issues, one additional issue has been raised that is inh

(different values have been used ranging from 12 to 200

been identified for resolution?

ntified issues) are/can be resolved without additional datentative safety assessment. If additional data are neede

l insufficient data announcement.

included in the

ientific Committee

Consumer Safety

ew of tricosan

re provided for your

eferences are a mix

in the next

, in part because of

t literature (in theeither productive

studies! The

an sourcing and

; and potential for

rent to the overall

mg/kg/d). Are

a, then it appears, they should be

CIR Panel Book Page 1



History ‐ Triclosan

At the request of CFSAN, the CIR Expert Panel placed triclosan on its high‐priority list in June of 2009.

A scientific literature review was issued April 9, 2010.




Search Strategy for Triclosan None. When it was discovered that multiple reviews of the available literature were already available and that key questions regarding the safety of triclosan already had been identified by FDA and others, the CIR literature review primarily was based on those secondary references. Since that approach was adopted, additional reviews have appeared on a regular basis and appear to present updated information, but a comparison of the reference lists continues to document that everyone is reviewing the same information. One study was cited by the European Commission in their analysis of public input on their 2009 opinion on triclosan safety that was not listed in their bibliography, but we contacted the authors at the USDA and obtained a copy of that publication (antimicrobial resistance study).




Report



Draft Report

Triclosan as used in Cosmetics

August 30, 2010

The 2010 Cosmetic Ingredient Review Expert Panel members are: Chairman, Wilma F. Bergfeld, M.D., F.A.C.P.;Donald V. Belsito, M.D.; Curtis D. Klaassen, Ph.D.; Daniel C. Liebler, Ph.D.; Ronald A Hill, Ph.D. James G.Marks, Jr., M.D.; Ronald C. Shank, Ph.D.; Thomas J. Slaga, Ph.D.; and Paul W. Snyder, D.V.M., Ph.D. The CIR

Director is F. Alan Andersen, Ph.D. This report was prepared by F. Alan Andersen, Ph.D., Director .

© Cosmetic Ingredient Review 1101 17

th Street, NW, Suite 412 " Washington, DC 20036-4702 " ph 202.331.0651 " fax 202.331.0088 "

[email protected]




Introduction1

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Triclosan is a chlorinated aromatic compound with functional groups representative of both phenols and

ethers. It is widely used in a variety of products, and including those regulated by the U.S. Environmental

Protection Agency (EPA) and Food and Drug Administration (FDA). EPA-regulated products include

those with a wide variety of antimicrobial uses (e.g., as a material preservative in a wide variety of

consumer, commercial, institutional, and industrial applications), while FDA-regulated products include

hand and body soaps, toothpastes, deodorants, laundry detergents, fabric softeners, facial tissues,

mouthwashes, antiseptics for wound care, and medical devices.

In April, 2010, the Cosmetic Ingredient Review (CIR) issued a scientific literature review of published and

unpublished data relevant to assessing the safety of triclosan in cosmetics. This literature review identified

a number of existing assessments of triclosan and provided information that was largely summarized from

those sources.

Since that time, three additional significant reviews of triclosan have been added. One is a critical review

of the experimental data and development of margins of safety for consumer products published in Critical

Reviews in Toxicology (Rodricks et al. 2010). Another other is European Commission’s Scientific

Committee on Consumer Products opinion on triclosan (European Commission 2009). And finally, the

Scientific Committee on Consumer Safety (same committee, new name) issued its opinion on triclosan

antimicrobial resistance (European Commission 2010).

Comments were received during the open public comment period. In addition, an unpublished

investigation of potential endocrine activity of triclosan was received (Environ International Corporation

2010).

Although it was suggested in comments on the literature review that more original literature citations

could/should be incorporated, the newly available published and unpublished reviews make it clear that all

of the available data have been captured and that the only differences are in data interpretation and

assumptions that are made. Creating another 63 page (Rodricks et al. 2010) or 136 page (Scientific

Committee on Consumer Products of the European Commission 2009) document would be redundant. Key

studies have been added, however, and a new section is now included that presents the risk assessment

approaches used by Rodricks et al. (2010) and the SCCP (European Commission 2009).

This draft safety assessment will broadly summarize the available data and focus on resolving those

questions critical to a determination of safety.

Regulation of Triclosan

FDA’s Center for Drug Evaluation and Research (CDER) regulates triclosan as a drug, including personal

care products with antibacterial/antimicrobial claims; and FDA’s Center for Devices and Radiological

Health (CDRH) is responsible for regulation of devices that may contain triclosan for

antibacterial/antimicrobial purposes. As defined by FDA, an antimicrobial (active) ingredient is "a

compound or substance that kills microorganisms or prevents or inhibits their growth and reproduction and

contributes to the claimed effects of the product in which it is included," and an antimicrobial preservative

(inactive) ingredient is defined as "a compound or substance that kills microorganisms or prevents or

inhibits their growth and reproduction and is included in a product formulation only at a concentration

sufficient to prevent spoilage or prevent growth of inadvertently added microorganisms, but does not

contribute to the claimed effects of the product to which it is added." A topical antimicrobial agent is

defined, in part, as "an antiseptic-containing drug product applied topically to the skin to help prevent




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infection in minor cuts, scrapes, and burns." Table 1 identifies triclosan-containing products regulated by

FDA.

EPA’s Office of Prevention, Pesticides and Toxic Substances regulates triclosan when used as an

antimicrobial (whether as a bacteriostat, fungistat, mildewstat, deodorizer and material preservative).

International health authorities in the European Union, Canada, Japan, Australia, and Norway regulate

triclosan in cosmetics and other products.

Tricolsan in Cosmetics

In the EU and other countries, antimicrobial and antiseptic products may be considered to be cosmetics,

and, as such, are controlled under cosmetic regulations which may not require pre-clearance or pre-market

approval of active ingredients. In Japan, antimicrobial and antiseptic agents may be regarded as drugs

subject to pre-approval. In Europe, Canada and Australia, the use of triclosan in cosmetics is limited to a

maximum concentration of 0.3%; in Japan, triclosan in cosmetics is limited to a maximum concentration of

0.1%, and Norway has stated that the use of triclosan in cosmetics should be limited, but no maximum

concentration is given. FDA’s Center for Food Safety and Applied Nutrition (CFSAN) is responsible for

the regulation of triclosan in cosmetics. CFSAN has asked CIR to undertake a review of the safety of

triclosan in cosmetics.

As given in the International Cosmetic Ingredient Dictionary and Handbook 1, triclosan may function in

cosmetic formulations as a cosmetic biocide, deodorant agent, or preservative.

Cosmetic Biocides are ingredients used in cosmetic products to help cleanse the skin or prevent odor by

inhibiting the growth of, or destroying microorganisms, such as bacteria, fungi or yeast. Cosmetic biocides

may be cidal or static. Cidal agents kill microbiota and act as disinfectants. Static agents inhibit the growth

of microorganisms but do not kill them. Ingredients used primarily for the protection of products against

contamination are found in the listing of Preservatives. Ingredients used as active ingredients in OTC drug

products which are intended to kill bacteria, fungi or yeast in order to treat, prevent or mitigate diseases are

included in the listing of Antimicrobial Agents.

Deodorants are ingredients that reduce or eliminate unpleasant odor and protect against the formation of

malodor on body surfaces. Absorbents can act as deodorants if they have the ability to absorb malodorous

chemicals. Also, chemical reactions can be used to destroy the malodorous substance in selected cases.

Perfumes and the like can be used to mask the perception of malodor by the process of reodorization.

Unpleasant odors also may be the result of microbiological activity. Thus, Cosmetic Biocides are

ingredients frequently used in skin-surface deodorants.

Preservatives are ingredients which prevent or retard microbial growth and thus protect cosmetic products

from spoilage. Cosmetic products may support the growth of microorganisms. The use of preservatives is

required to prevent product damage caused by microorganisms and to protect the product from inadvertent

contamination by the consumer during use. The use of more than one preservative can sometimes increaseefficacy due to synergism. Ingredients used to protect products against oxidative damage are classified as

Antioxidants.

Government Body Triclosan Assessments


http://online.personalcarecouncil.org/jsp/IngredInfoDropResultPage.jsp?preference=50&IngredInfoList=14






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In preparing this report, CIR relied extensively on triclosan reviews available from various governmental

sources as an alternative strategy to summarizing the large volume of original literature2-7:

European Centre for Disease Prevention and Control (ECDC), European Food Safety Authority3

(EFSA), European Medicines Agency (EMEA), Scientific Committee on Emerging and Newly4

Identified Health Risks (SCENIHR). “Joint Opinion on Antimicrobial Resistance (AMR) Focused on5

Zoonotic Infections.” October 2009.6

http://ec.europa.eu/health/ph_risk/committees/04_scenihr/docs/scenihr_o_026.pdf 7

European Commission Directorate-General for Health & Consumers. Scientific Committee on

Emerging and Newly Identified Health Risks (SCENIHR). Assessment of the Antibiotic Resistance

Effects of Biocides. January 2009.

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http://ec.europa.eu/health/ph_risk/committees/04_scenihr/docs/scenihr_o_021.pdf 11

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Australian Government Department of Health and Ageing (NICNAS). Priority Existing Chemical

Assessment Report No. 30 – Triclosan. January 2009.

http://www.nicnas.gov.au/Publications/CAR/PEC/Drafts/Triclosan.asp .14

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United States Environmental Protection Agency (EPA). Office of Prevention, Pesticides and Toxic

Substances. Reregistration Eligibility Decision (RED) for Triclosan, List B, Case No. 2340. EPA 739-RO-8009. September 2008. http://www.epa.gov/oppsrrd1/REDs/2340red.pdf 17

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National Toxicology Program. FDA Nomination Profile - Triclosan [CAS 3380-34-5]. Supporting

Information for Toxicological Evaluation by the National Toxicology Program. July 2008.

http://ntp.niehs.nih.gov/ntp/htdocs/Chem_Background/ExSumPdf/triclosan_508.pdf .20

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United States Environmental Protection Agency (EPA). Memorandum. January 4, 2008. Triclosan:

Report of the Cancer Assessment Review Committee. PC Code: 054901.

http://www.epa.gov/pesticides/chemical/foia/cleared-reviews/reviews/054901/054901-2008-01-04a.pdf 23

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Since these summaries of the available data were reviewed, a major review of triclosan safety has been

published in Critical Reviews in toxicology (Rodricks et al. 2010). In addition, the European

Commission’s SCCP opinion on triclosan (European Commission 2009) was reviewed and the Scientific

Committee on Consumer Safety (same committee, new name) issued its recent opinion on triclosan

antimicrobial resistance (European Commission 2010). Information from the critical review and the two

European Commission opinions has been added to provide added detail or information not included in the

above governmental bodies reviews. In addition, a new section was added to present exposure assessments

and margin-of-safety determinations.

Report structure

Because this document departs from the approaches that CIR has used in the past to initiate a safety

assessment of cosmetic ingredients, a brief overview of what is included and why is appropriate.

Section I addresses the relevant issues for triclosan as used in cosmetics.

Section II presents technical names and synonyms, physicochemical properties, information on methods of

manufacturing, chemistry methods for identification and analysis, information on impurities and

photostability.


http://ec.europa.eu/health/ph_risk/committees/04_scenihr/docs/scenihr_o_026.pdf


http://www.nicnas.gov.au/Publications/CAR/PEC/Drafts/Triclosan.asp

http://www.epa.gov/oppsrrd1/REDs/2340red.pdf

http://ntp.niehs.nih.gov/ntp/htdocs/Chem_Background/ExSumPdf/triclosan_508.pdf

http://www.epa.gov/pesticides/chemical/foia/cleared-reviews/reviews/054901/054901-2008-01-04a.pdf




http://www.nicnas.gov.au/Publications/CAR/PEC/Drafts/Triclosan.asp





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Section III provides information on the extent of use of triclosan in cosmetics based on information

provided by the industry to the FDA’s Voluntary Cosmetic Registration Program (VCRP). Use

concentrations based on a survey conducted by the Personal Care Products Council also are provided.

Section IV provides a limited overview of triclosan’s absorption/toxicokinetics, distribution, metabolism,

and excretion.

Section V presents an overview of assessments that have been made on triclosan’s potential toxicological

hazards, including endocrine hazards.

Section VI provides information on triclosan’s putative mechanisms for the inhibition of bacterial growth

and presents the key arguments that have been raised about triclosan’s potential for causing antibiotic and

antibacterial resistance.

New Section VII includes rationales for benchmark doses and/or no-observable-adverse-effects-levels,

consumer exposures, and margins-of-safety for triclosan in consumer products.

Finally, Section VIII summarizes and integrates information in the preceding sections.

I. Issues to be resolved in safety substantiation of triclosan as used in cosmetics.14

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1. Triclosan exposure.

Issue: uses of triclosan in OTC drugs may present different exposure scenarios compared to use in

cosmetics.

Current status: information on the number of personal care products that contain triclosan, and at what use

concentration, is now available. Various analyses of the use of those products also are available.

2 Triclosan sourcing and dioxin impurities.

Issue: triclosan imported from India and China reportedly may contain dioxin compounds.

Current status: further information on sourcing was not available, although impurities data do indicate low

levels of dioxins. Limits on the levels of dioxin compounds in triclosan as supplied for use in cosmeticscould be established.

3. Photostability and dioxin photoproducts

Issue: triclosan applied to the skin may photodegrade to dioxin compounds on exposure to light.

Current status: no further data were provided. Empirically, this question may be addressed by the available

data demonstrating the absence of phototoxicity in animal tests.

4. Carcinogenicity

Issue: data from one mouse carcinogenicity study did suggest a statistically significant increase in liver

carcinomas and adenomas as a function of dose, above a threshold level.

Current status: the mode of action for these mouse liver tumors appears to be by a mechanism that is not

relevant to human health considerations. When NTP carcinogenicity data are available, they should be

considered.




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5. Endocrine disruption

Issue: triclosan may bind to estrogen and/or androgen receptors and thus act as an endocrine disruptor.

Current status: a mix of endpoints that may be considered as endocrine effects have been studied. These

data include studies that found no effect, and other studies suggesting weak estrogenic and androgenic

effects, and antiestrogenic and antiandrogenic effects, suggesting a mix of endocrine action.

6. Potential for bacterial resistance

Issue: any antibiotic/antimicrobial agent potentially can be a selective agent for resistance in target

organisms.

Current status: while bacterial strains with resistance to triclosan can be developed in vitro, this

phenomenon is not seen in surveillance studies of organisms in most use situations. While some data are

available demonstrating that emergence of triclosan resistance is accompanied by resistance to common

antibiotics, other studies failed to find such a link.

II. Chemistry13

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Definition and Structure

The International Cosmetic Ingredient Dictionary and Handbook has established triclosan as theInternational Nomenclature Cosmetic Ingredient (INCI) name (to be used in cosmetic product labeling) for

the substituted organic ether that conforms to the structure shown in Figure 1.1 The National Library of

Medicine’s ChemIDPlus website at http://chem.sis.nlm.nih.gov/chemidplus/ (enter Triclosan) shows the

same structure and provides other data sources, e.g. the European chemical Substances Information System,

and the FDA Drug Database.8

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In addition to being an INCI name, triclosan also is a INN name (International Nonproprietary Names for

Pharmaceutical Substances, WHO). Although other CAS numbers have been used previously for triclosan,

the current CAS number is 3380-34-5 and the EINECS number is 222-182-2.

As given in the International Cosmetic Ingredient Dictionary and Handbook 1, triclosan is sold under a

variety of trade names and trade name mixtures that contain triclosan (supplier name given in parentheses):

Trade names

• AEC Triclosan (A & E Connock)

• Amicare 100 (Cosmetic Rheologies)

• Dekaben TC (Dekker)

• Irgacare MP (Ciba Specialty Chemicals)

• Irgasan DP (Ciba Specialty Chemicals)

• Jeechem Triclosan (Jeen Int. Corp.)

• Mackstat TCN (McIntyre) – note that this is actually triclosan in a solvent.

• Oletron (Sino Lion)

• OriStar TC (Orient Stars)

• Rita Triclosan (Rita)

• Triclosan (Kumar Organic Products)

• Triclosan-PC (Pretameen)

Trade name mixtures


http://chem.sis.nlm.nih.gov/chemidplus/




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• Custoblend BAT (Custom Ingredients)

• Elestab 4121 (Laboratories Serobiologiques)

• Irgasan PG-60 (Ciba Specialty Chemicals)

• Lipobead Blue-T (Lipo)

• Lipo CD-TC (Lipo)

• Miracare AB-33 (Rhodia)

• Rovisome Acne (Rovi GMBH)

The SCCP (European Commission 2009) also lists Irgacare CF100 and Irgacide LP10 as trade names.

Because triclosan is a powder, any material supplied as a liquid must be a mixture with a solvent. Also,

trade name mixtures will contain chemicals in addition to triclosan, and the safety of these additional

chemicals may not have been determined.

Physical and Chemical Properties

Triclosan’s physical and chemical properties are presented in Table 2.

Method of ManufactureAs given in the 6th edition of Ullmann's Encyclopedia of Industrial Chemistry,10 triclosan is produced by

treatment of 2,4,4′-trichloro-2′-methoxydiphenyl ether with aluminum chloride in benzene under reflux.

Conversion to chlorinated dibenzo- p-dioxins (see Impurities below and Figure 2) can occur under extreme

conditions such as high alkalinity and heat. The type and purity of the starting materials in the synthesis of

triclosan influenced the extent of contamination by the impurities dioxins and dibenzofurans.4

Methods of detection.

Triclosan may be separated using using high performance liquid chromatography,4 and detected by infrared

and/or UV (peak absorption at 281 nm)52 spectroscopy. Gas chromatography/mass spectroscopy

methodology has a detection limit for triclosan of 0.5 ng/ml.53

Impurities

Commercial grade triclosan is reported to be >99% pure (w/w) as the powder, and 10 to <20% (w/v) pure

as a liquid solution.4 Technical grade triclosan produced by Ciba and Harmet/Vivimed is >99.0% and

99.9% pure, respectively.7 Trace level impurities identified by the US Pharmacopoeia (USP) include

mono- and di-chlorophenols, as well as di-, tri-, and tetra-chlorodibenzo-p dioxins and di-, tri-, and tetra

chlorodibenzofurans.11

Menoutis and Parisi tested samples of triclosan from India and China for the presence of dioxins.12 Six

samples of triclosan, each of which were manufactured by a different producer in India or China (5 samples

and 1 sample, respectively, from each country), were analyzed for the presence of 2,3,7,8-

tetrachlorodibenzo- p-dioxin (TCDD) and 2,3,7,8-tetrachlorodibenzofuran (TCDF). All six samplescontained TCDD in excess of 1 pg/g, and 4 of the six samples contained TCDF in excess of 1 pg/g. TCDD

and TCDF ranged from 17.2 pg/g to 1712.0 pg/g, and 0.43 pg/g to 207.30 pg/g, respectively, as shown in

Table 3. The authors suggested that the presence of these two trace impurities may be due to the quality or

purity of the starting material, the particular synthetic process, or the inability to tightly control physical

synthetic parameters.




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Ciba Specialty Chemicals (now part of BASF) has reported that Irgasan® DP 300 (which Ciba

manufactures and distributes for topical use, specifically) and Irgasan® MP, meet USP’s requirements11 (see

Table 4), but that generic triclosan made by other manufacturers does not necessarily comply with USP

specifications.13

As noted earlier, impurities in triclosan that may be present in trace amounts include dioxins, and which are

limited or not allowed by the U.S. Pharmacopoeia (USP). In addition, the government of Canada has

established limits on dioxins. All these limits are presented in the Table 4. Triclosan is not on the work

program of either the British Pharmacopoeia Commission or the European Pharmacopoeia Commission.14

FDA has stated that it is unaware of the purity, identity and concentration of impurities in triclosan used in

cosmetics, or the sources of triclosan that is used in cosmetic formulations in the US.15 Information on

impurities from all triclosan suppliers would be useful in evaluating the need to establish limits for

allowable impurities in cosmetic-grade triclosan.

Chemical Reactivity

According to Rule et al.,16 chloroform may be produced if a soap containing triclosan comes into contact

with chlorinated water. Two dish soaps, one containing triclosan (at 1.4 mg triclosan/g soap) and one

without, were added to chlorinated water at a concentration of 0.25 g/L. The measured chloroform levelwas 15 µg/L after 5 min and 49 µg/L after 120 min for the triclosan-containing soap. The chloroform

levels for the non-triclosan formulation were near the detection limit.

Photostability

In FDA’s nomination of triclosan for study by the National Toxicology Program,6 the agency argued that

the level of dichlorodibenzo- p-dioxins in the environment following photodecomposition of triclosan, and

the levels of dichlorodibenzo-p-dioxins on skin following photodecomposition of topically applied triclosan

have not been established.

Lores et al. (2005)17 reported that, under artificial conditions, triclosan can photodegrade to 2,7- and 2,8-

dichlorodibenzo- p-dioxin (2,7/2,8-DCDD). In addition, 2,4-dichlorophenol (DCP), which is not a dioxin,

has been identified as a major degradation product under artificial conditions - 93.8-96.6% of the applied

triclosan degrades to DCP within 240 minutes post-treatment.18

Since the pKa for triclosan is around pH 8.1, at physiological pH, the phenolic form (shown in Figure 1)

would predominate while at pH of 9, for example, the phenolate form (shown in Figure 3) would

predominate. NICNAS stated that the phenolic form of triclosan is relatively photostable, whereas the

phenolate form is more photodegradable.

NICNAS4 included the proposal that triclosan photolysis products would include the three permutations of

dichloro compounds; a dihydroxy coumpound (2,4’-dichloro-2’,4-dihydroxydiphenyl ether), which could

further degrade to a monochloro compound, 4-chloro-2,4’-dihydroxydiphenyl ether; or 4-chloro-2-

hydroxyphenol, which is closely related to the DCP photodegradation product noted by above.

Overall, some information suggests that photodegradation, likely by UV light, may produce dioxin

compounds, but other sources have postulated other photodegradation products that are not dioxin




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compounds. Since the photodegradation phenomenon appears to be pH dependent, one useful group of

data would be pH values for representative cosmetic product categories that contain triclosan.

III. Extent of use and use concentrations for triclosan in cosmetics.3

According to information supplied to FDA by industry as part of the Voluntary Cosmetic Registration

Program (VCRP),19 personal hygiene products is the category with the greatest number of products

containing triclosan (226 triclosan-containing products, which represents ~7% of products in this category

overall). Since FDA does not verify labelers’ product status with regards to its VCRP database,15

it may bethat some of these products are marketed with antibacterial claims, and could be considered drug products

or both drug products and cosmetic products. The skin care products category ranks second, with 162

triclosan-containing products, which represents ~2% of all products in this category. FDA VCRP data for

triclosan are given in Table 5. FDA’s VCRP is, as the name of the program implies, “voluntary.” As a

result, these data cannot be regarded as complete.

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A Personal Care Products Council (“Council”) survey reported that triclosan is used in cosmetics at

concentrations ranging from 0.01 to 0.3%.20 It is not known that every company using triclosan is

represented by these data.18 Use concentration survey responses are given in Table 5, as a function of

product category.

To interpret the data in Table 5, consider that, of all baby shampoos reported (total of 56), only 1 contains

triclosan (~2% of all baby shampoos reported), or conversely, the vast majority do not, and the situation is

similar for the baby lotions, powders, and creams category. While uses of triclosan were reported to FDA

under the VCRP for each of these categories, no use concentrations were provided in the industry survey

for these categories. And no uses of triclosan or use concentrations were reported for the 143 products in

the “other” baby products category - usually this row would not be included in Table 5 because there are no

data to provide, but in this case it was not deleted because information that there is a product category in

which there are no products containing triclosan may be important information.

Rodricks et al. (2010) reported use concentrations that are consistent with the data from the Council survey,

except that a use concentration range up to 0.45% was reported for a liquid hand soap. The SCCP(European Commission 2009) reported use concentrations up to 0.3%.

Table 5 presents all of the cosmetic product categories, so that a reader may see all categories in which

triclosan is used and at what levels (depending on availability of those data), as well as the cosmetic

product categories in which triclosan is not used.

According to the Skin Deep Cosmetics Safety Database21 that lists OTC drug and cosmetic products for

which triclosan is listed as an ingredient on the product label, a total of 938 products contain triclosan, one

of which is in a cosmetic product categories for which no uses were reported to FDA’s VCRP. For

example, the label for Oscar Blandi Pronto Dry Shampoo (presumably a shampoo) lists Triclosan as an

ingredient, yet no shampoos were reported to the VCRP containing triclosan. There was one babyshampoo reported, but Oscar Blandi Pronto Dry Shampoo does not appear to be a baby shampoo.

Triclosan-containing rinse-off and leave-on cosmetics uses may include products that result in triclosan

exposure by the dermal, inhalation, and oral routes. Dermal exposure appears to include rinse-off and

leave-on cosmetics applied to adults, as well as to children.


http://www.cosmeticsdatabase.com/product/57856/Oscar_Blandi_Pronto_Dry_Shampoo/






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Cosmetic aerosols

Safety of inhaled aerosols depends on the ingredient, the concentration, the duration of the exposure and

where they are deposited within the respiratory system.22 The site of deposition is associated most with the

particle size and density of the particle being inhaled. In general, the smaller the particle, the further into

the respiratory tree the particle will deposit and the greater the impact on the respiratory system.

The parameter most closely associated with this regional deposition is the aerodynamic diameter, da,

defined as the diameter of a sphere of unit density possessing the same terminal settling velocity as the particle in question. In humans, particles with an aerodynamic diameter of ≤ 10 µm are respirable.

Particles with a da from 0.1 – 10 µm settle in the upper respiratory tract and particles with a da < 0.1 µm

settle in the lower respiratory tract. 23,24

Particle diameters of 60-80 µm and ≥80 µm have been reported for anhydrous hair sprays and pump

hairsprays, respectively.25 In practice, aerosols should have at least 99% of their particle diameters in the 10

– 110 µm range and the mean particle diameter in a typical aerosol spray has been reported as ~38 µm.26

Therefore, most aerosol particles are deposited in the nasopharyngeal region and are not respirable.

IV. Absorption/toxicokinetics, distribution, metabolism and excretion15

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NICNAS4 presented a review of triclosan’s absorption/toxicokinetics, distribution, metabolism and

excretion. These data were supplemented with data reviewed by FDA, 6 specifically for data that NICNAS4

did not address, or had discounted because of shortcomings in reporting.

Absorption/Toxicokinetics

Most reviews have suggested that triclosan is slowly and not extensively absorbed by the dermal route,

consistent with its low water solubility and log Po/w of 4.8, but is rapidly and well absorbed by the oral

route.

Rodricks et al. (2010) suggested that dermal absorption would likely be <10%, but that oral absorption

would be complete. In human subjects, for example, daily use of triclosan-containing toothpaste for up to

65 weeks resulted in increased blood levels compared to pre-use levels, but those increased levels remained

steady and returned to baseline after use. Using full thickness human skin, total absorption of triclosan was

vehicle specific, with dishwashing liquid at 12%, water/oil emulsion at 11.3%, deodorant at 7.65%, and

soap solution at 7.2%.

These same dermal absorption figures were reported in the SCCP opinion on triclosan (European

Commission 2009).

Distribution

Triclosan measured in rodent radioactivity studies (following oral and dermal exposures) indicate

distribution at highest levels to the liver, lung, kidney, gastrointestinal tract, and gall bladder.4

Rodricks et al. (2010) suggested that differences in distribution between mice, rats, and hamsters (plasma

levels are higher than liver or kidney levels in rats and hamsters, but not mice) implies that triclosan can

accumulate in the mouse liver.




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Oral and dermal routes (humans and rodents): Triclosan absorbed from the gastrointestinal tract undergoes

extensive first-pass metabolism, which primarily involves glucuronide and sulfate conjugation. In both

humans and rodents, at high triclosan plasma concentrations, metabolism shifts from the generation of

predominantly glucuronide conjugates to sulfate-conjugates. The bioavailability of unconjugated triclosan

may be limited after oral exposure because of triclosan’s extensive first-pass metabolism. Triclosan is also

metabolized to its glucuronide and sulfate conjugates by the skin.4 FDA concluded that > 90% of absorbed

triclosan is metabolized.6

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Rodricks et al. (2010) noted that the glucuronide metabolite predominates in humans while the sulfate

conjugate is the dominant metabolite in mice.

The SCCP (European Commission 2009) also emphasized the extensive first-pass metabolism and the

almost-total conversion to glucuronide and sulfate metabolites. Based on results from oral studies (e.g.,

toothpaste use) and dermal studies (e.g., washing with soap), there was no evidence of accumulation of

triclosan in the human body.

Excretion

Oral and dermal routes (humans and rodents): Triclosan glucuronide is predominantly excreted in the

urine, and triclosan is predominantly excreted in the feces. Triclosan that is administered orally anddermally is excreted in greater concentrations in the urine than in the feces in humans, hamsters, rabbits,

and monkey. In rats, mice, and dog, the reverse is true. Up to 87% of triclosan that is administered to

humans (by an unspecified route) is excreted in the urine, most of it within 72 h after dose.4

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Rodricks et al. (2010) noted that elimination half-lives following repeated dermal application of triclosan

(1.4 to 2.1 days) are greater than those following oral administration (10 to 20 hours).

Biomonitoring Data

According to Rodricks et al. (2010), several studies have reported triclosan in plasma and urine in the

general population and in human breast milk in nursing mothers. Triclosan amounts in breast milk were

reported to range from <20 to 300 μg/kg lipid in one study and <5 to 2100 μg/kg lipid in another. In astudy that compared triclosan levels in women who used triclosan-containing products with those who did

not, levels in breast milk were 0.022 to 0.95 μg/kg lipid compared to 0.018 to 0.35 μg/kg lipid,

respectively.

The largest bio-monitoring study was conducted as a subset of the National Health and Nutrition

Examination Survey (NHANES) in which urine samples were taken from a random ⅓ of the 9643 subjects

yielding data on 2514 individuals (Calafat et al. 2008). For the entire sample, the geometric mean triclosan

level in urine was 13 μg/l. There were age differences in the findings as well as sex differences for urine

concentrations, as shown in Table 6.

V. Toxicology/Safety36

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This section presents an overview of studies performed in experimental animals models or in vitro systems

(acute toxicity, dermal and eye irritation, phototoxicity, sensitization, repeat dose toxicity, reproductive

toxicity, endocrine disruption, genotoxicity, and carcinogenicity), as well as in humans (skin irritation and

sensitization).




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Acute toxicity

Triclosan has low acute toxicity by all evaluated routes and in all evaluated species.6,7 Oral rat and mouse

LD50 values were > 3700 mg/kg. Rabbit dermal LD50 values were > 9000 mg/kg and the rabbit inhalation

LC50 is > 0.15 mg/L. The rat subcutaneous and intraperitoneal, and intravenous LD50 values were > 14,700

mg/kg, >1090 mg/kg, and 29 mg/kg. The dietary NOAEL for triclosan in baboons was 30 mg/kg, with a

LOAEL of 100 mg/kg/day, a dose at which clinical signs were observed, such as vomiting, failure to eat,

and diarrhea.

Dermal and eye irritation; phototoxicity, respiratory irritation, sensitization

In rabbits, triclosan was a moderate dermal irritant (Primary Irritation Index score of 3.5 @ 72 hours), as

well as a moderate eye irritant.7 Triclosan in various formulations at < 0.25%, when tested dermally in a

rabbit primary dermal irritation test and acute dermal lethality test did not cause dermal toxicity.29 EPA7 did

not consider triclosan a sensitizer in guinea pigs, although Australia4 considered it a very weak sensitizer in

the same model. Triclosan was not a phototoxicant in guinea pigs.4 NICNAS considered triclosan to be a

respiratory irritant.4

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Most recently, Rodricks et al. (2010) reviewed the available dermal irritation and sensitization studies.

Dermal irritation was concentration dependent. For example, a single application of triclosan at 0.3% was

not irritating in any animal species, but dermal concentrations of 5% were irritating to guinea pig skin and

ocular concentrations of 1 to 100% produced reversible eye irritation in rabbits. Repeated dosing dermal

studies (14-day) have consistently found a threshold of around 1.5% for irritation. Sensitization studies

were negative in guinea pigs at 0.1% when administered subcutaneously, or topically at concentration up to

10%. Triclosan at concentrations up to 1% were not photosensitizers in guinea pigs, mice, or pigs with

irradiation in either the UVA, UVB, or UVC region.

The SCCP (European Commission 2009) also reviewed the available photosensitization data in guinea

pigs, mice, and pigs and, while noting that all of the available studies predated good laboratory practices,

suggested that there was no evidence for photosensitization.

Repeat dose toxicity

Triclosan repeat dose toxicity has been evaluated in the baboon and hamster (oral route), rat (oral, and

inhalation routes), mouse (dermal route) and rabbit (dermal). Triclosan NOAELs based on local irritation

effects tended to be < 10 mg/kg/day by all routes, except inhalation, which had a reported No-Observed-

Adverse-Effect-Concentration (NOAEC) of 5 x 10-5 mg/m3. LOAELs and NOAELs based on systemic

toxicity tend to be <1000 mg/kg/day, with no obvious common toxicity among studies and species.

NICNAS,4 EPA,5,7 and FDA6 toxicology data summaries are presented in Table 8. NICNAS4 or FDA6,19

summary statements were not included if such statements did not provide duration of exposure or

information on doses, or were fundamentally flawed (e.g., LOAEL lower than the NOAEL). Table 7 is

organized hierarchically by route of exposure, duration of dosing (subacute → subchronic → chronic),

species (monkey → rat/mouse → rabbit). Unless otherwise noted, NOAEL units were not standardized.

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Rodricks et al. (2010) summarized findings from repeated dose dermal exposures of triclosan in propylene

glycol or acetone vehicles using CD-1 mice (doses from10 to 200 mg/kg/day) and Crl:CD BR rats (doses

from 1.2 to 24 mg/kg/day). Responses varied as a function of vehicle, dose, species, and sex of the

exposed mice. For example, in mice, liver weights were increased in males at all doses >10 mg/kg/day,

independent of vehicle, but only at 200 mg/kg/day in females for the propylene glycol vehicle exposures.

Pale foci were noted in the livers of male mice from the 100 and 200 mg/kg/day groups with both vehicles,




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but not in females. No significant changes in liver weights were reported in rats, nor were there any effects

seen on macroscopic or microscopic examination, in either sex.

Genotoxicity

Trisclosan has been evaluated in a number of standard (and other) genotoxicity assays, including bacterial

reverse mutation tests, in vitro mammalian cell gene mutation test, and in vitro mammalian chromosome

aberration tests, a mammalian bone marrow chromosomal aberration test, and an unscheduled DNA

synthesis assay in mammalian cells in culture. As FDA6

noted: the preponderance of data suggested thattriclosan is not genotoxic. NICNAS4 drew a similar conclusion. EPA5 provided a detailed review of seven

genotoxicity tests. It concluded that each test, except one (an in vitro cytogenetic assay with Chinese

hamster lung fibroblasts), was negative. Therefore, the consensus on the weight of evidence on triclosan’s

genotoxicity potential is that it is not genotoxic.

Carcinogenicity

NICNAS (2009)4 and EPA7 concluded that triclosan was not carcinogenic based on the available data.

FDA, however, has concluded that the available data are not adequate to resolve the question of triclosan

carcinogenicity via the dermal route of exposure seen in skin cleansing preparations.6

EPA7 specifically evaluated rat and hamster oral chronic toxicity/carcinogenicity studies and concluded that

triclosan exhibited no carcinogenic potential in rats at < 3000 ppm and in hamsters at < 250 mg/kg/day.

However, EPA reviewed a mouse oral chronic/carcinogenicity bioassay and found it positive for

carcinogenicity based on an increased incidence of liver neoplasms in male and female mice at

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mg/kg/day.7 Nevertheless, EPA concluded that this study did

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not support triclosan carcinogenicity, and

that triclosan is “not likely to be carcinogenic in humans”. This conclusion was based on the weight of

evidence that supports activation of peroxisome proliferator activated receptor alpha (PPAR α) as the

primary mode of action for triclosan-induced hepatocarcinogenesis in mice. Also, EPA stated that, while

the data did not support either a mutagenic mode or cytotoxic mode of action, that the mode of action for

liver tumors in mice is theoretically plausible in humans. Based on differences in the PPAR α responses in

humans compared to mice, however, EPA suggested that such a mode of action was unlikely.

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In referring triclosan to the NTP for study, FDA specifically commented on oral toxicity data using albino

rats submitted to the agency in 1977, suggesting that the presence of test material in control animals

invalidated the results and on an oral rat study conducted in 1986, suggesting that the study was inadequate

based on a high rate of mortality, absence of significant body weight differences between treated and

control animals, and the presence of hepatocellular lesions not consistent with the morbidity/mortality.6 In

spite of the agency describing the latter study as inadequate, one FDA reviewer concluded that Triclosan

was oncogenic at 3,000 ppm at 104 weeks. In 1999, FDA reviewed another carcinogenicity study

(hamsters) and apparently formed no conclusion on the merits of the study because the sponsor did not

respond to the Agency’s request for histopathology slides of kidneys, liver, lungs, adrenals and all tumorsfrom all animals on study for review.

FDA,6 in its presentation of the rationale for NTP study of triclosan, also suggested that the only available

dermal toxicity data (90 day dermal rat study) could be interpreted to suggest dose-dependent abnormalities

which need subsequent study with a 2-year dermal carcinogenicity bioassay.




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Rodricks et al. (2010) reviewed chronic toxicity studies using rats, hamsters, and mice in which the

incidence of tumors was evaluated. Rats were fed triclosan in the diet at 0, 300, 1000, and 3000 ppm for up

to 104 weeks, with an additional group at 6000 ppm for 52 weeks. No evidence of tumors or preneoplastic

lesions was found.

In its discussion of these data, the SCCP (European Commission 2009) noted that the exposures were

calculated to yield doses of 0, 12, 40, and 127 mg/kg/d for males and 0, 17, 56, and 190 mg/kg/d for

females. The additional dose for the 52-week animals was calculated to be 247 mg/kg/d for males and 422

mg/kg/d for females. The SCCP did not disagree that no evidence of tumors or preneoplastic lesion wasfound, but did determine that there were significant reductions in red blood cell counts in males and

females, including low-dose males at 104 weeks. Increases in mean corpuscular hemoglobin were

observed in mid- and high-dose females and in males at all doses. Other hematologic parameters were also

different from controls, but only in the high-dose groups.

The SCCP also noted decreased absolute and relative spleen weights in mid-dose females.

The SCCP considered that these hematotoxicity results and the spleen weight changes as indications of an

adverse effect and established the NOAEL from this study at 12 mg/kg/d. This is the calculated lowest

dose for male animals, but the SCCP did not comment on the red blood cell count changes or the increases

in mean corpuscular hemoglobin that were observed in low-dose males.

Rodricks et al. (2010) suggested that the statistically significant hematological changes were slight and

transient (red blood cell counts were down at 13, 26, and 52 weeks, but not at 78 or 104 weeks) and that

there were no other indications that the animals were anemic. These authors also noted an absence of

macroscopic or microscopic evidence of an effect on the hematopoietic system and no apparent effects on

homeostasis.

Since this 104-week rat study will likely be a focus of discussion, CIR will try to have the original

unpublished study available at the meeting.

Rodricks et al. described a study in which hamsters were fed triclosan in their diet at 0, 12, 75, or 250

mg/kg/day for 90-95 weeks. Deaths in male hamsters in the high-dose group were significantly higher than

in the control group. No evidence of liver damage was seen at any dose, but body weight gain was

significantly reduced and nephropathy was significantly increased in both sexes at the highest dose

compared to controls. In addition, hyperplasia in the fundic region of the stomach, abnormal

spermatogenic cells, reduced spermatozoa, and germ-cell depletion were noted in high-dose males.

The SCCP (European Commission 2009) review of these data also noted the positive findings in high-dose

hamsters and set the NOAEL at 75 mg/kg/d.

Rodricks et al. (2010) reviewed the study in which CD-1 mice were fed triclosan in the diet at doses of 0,

10, 30, 100, or 200 mg/kg/d for 6 months or 18 months. In the 18-month study, statistically significant

increases in liver adenomas and carcinomas were seen at several dose levels compared to controls as shown

in Table 8.

The SCCP (European Commission 2009) did note an increased incidence in liver tumors at doses of 30

mg/kg/d, and commented that triclosan is a peroxisome proliferator in mouse liver. The SCCP adenomas




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described dose-related increases in liver weights at 30, 100, and 200 mg/kg/d in males and females, and

hepatocyte hypertrophy in males at all doses. The LOAEL was established at 10 mg/kg/d.

While triclosan in the diet appears to be linked to the adenomas and carcinomas in the liver of the exposed

mice, the questions that arise from this finding are: (1) why did those tumors occur, and (2) is the finding

relevant to human health? For example, since mice accumulate triclosan in the liver and humans don’t,

might this explain the causation (high accumulated levels of triclosan) and be an adequate basis for

discounting the effect for human exposure?

Rodricks et al. (2010) used the International Programme on Chemical Safety (IPCS) framework (Boobis et

al. 2006) to assess the relevance of the mode of action (MOA) of tumor formation in the mouse study to

humans. This approach posits three questions:

1. Is the weight of evidence sufficient to establish an MOA in mice for tumor formation?

2. Is the MOA relevant to human health (i.e., can it even happen in humans)?

3. Even if the MOA can happen in humans, is the MOA inconsequential on the basis of quantitative

differences in either kinetic or dynamic factors between mice and humans?

The first step in addressing these questions, obviously, is to postulate the MOA of triclosan in the mouseliver that produces tumors. While hepatic tumors are the most common spontaneous tumors in mice, the

mouse liver is a frequent target of chemically induced tumors. MOAs for chemically induced liver tumors

include genome mutation in liver cells, or non-genotoxic gene activation/deactivation, and/or receptors.

As discussed earlier, the preponderance of evidence is that triclosan is not genotoxic, so the MOA is likely

non-genotoxic. Activation of peroxisome proliferator-activated receptors (PPARs) is a well-characterized,

non-genotoxic mechanism by which a cascade of events can lead to tumor formation. Three types of

PPARs have been identified: α, β/δ, and γ, with the α form expressed in the liver. In concept, a ligand

binds to a retinoid X-receptor (RXR) in the cytoplasm, is transported to the nucleus, where the combination

ligand/RXR binds to promoter sequences of peroxisome proliferation genes, activating PPAR α. That alters

the transcription of genes involved with peroxisome proliferation, apotosis, and lipid metabolism. Those

changes increase fatty acid β-oxidation which can lead to oxidative stress. In turn, increased stimulation of

nonparenchymal cells and inhibition of gap junction intercellular communication can occur. Increased cell

proliferation and decreased apotosis, leads to hyperplasia and hepatic tumors.

So, for the mice in the triclosan carcinogenicity study, is there evidence of triclosan-related PPAR α

activation, cell proliferation, fatty acid β-oxidation, etc? Rodricks et al. (2010) reviewed the available data

and concluded that there was no direct evidence of PPAR α activation, but there was evidence of triclosan-

related PPAR α-dependent up-regulation of CYP3A and CYP4A, testosterone hydroxylation, and lauric

acid 11-12 hydroxylation, and PPAR α-dependent expression of nonperoxisomal fatty acid metabolism

genes (cyanide-independent palmitoyl CoA oxidation). Peroxisome proliferation was supported by the

findings of triclosan-related hypertrophy due to an increase in the number and size of peroxisomes and an

increase in smooth endoplasmic reticulum. While there was no evidence of PPAR α-dependent expression

of cell-cycle growth and apotosis, there were triclosan dose-dependent increases in Proliferating Nuclear

Cell Antigen (PNCA) labeling index, indicative of perturbation of cell proliferation and/or apotosis.

Hepatocyte oxidative stress was suggested by the triclosan dose-related increases in lipofuscin in the

Kupffer cell region. Kupffer cell-mediated events were suggested by triclosan-related Kupffer-cell


http://en.wikipedia.org/wiki/Peroxisome_proliferator-activated_receptor_alpha





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activation. And finally, selective clonal expansion is suggested by the finding of triclosan-related hepatic

adenomas and carcinomas.

The authors considered the possibility that hepatic cytotoxicity could be the MOA. In concept, triclosan

would be cytotoxic, resulting in a hyperplastic response, during which hepatic cells with DNA damage

proliferate, produce preneoplastic foci, and then tumors. While cell proliferation was linked to triclosan

treatment, necrosis was not.

If PPAR α activation is the MOA of triclosan in mice, then how would it translate to a human health risk?Expression of PPAR α in the human liver is 1/10th of that in the mouse. A study of a known liver

carcinogen in mice containing the gene for human PPAR α compared to mice containing the normal mouse

gene produced no tumors in the mice containing the human gene and the expected tumors in the mice

containing the normal mouse gene. That said, activation of PPAR α and expression of peroxisomal genes

can occur in humans.

Is there something about how humans metabolize, distribute, and excrete triclosan that suggests the mouse

MOA would not be applicable? Certainly, excretion is different. In mice, triclosan is mostly excreted in

the feces as unchanged parent chemical. In humans, the primary excretion route is in the urine as the

glucuronide conjugate. Also, the method of excretion in mice supports the finding that triclosan can

accumulate in the mouse liver.

Overall, Rodricks et al. (2010) concluded that the hepatic tumors produced by a PPAR α activation MOA

are not relevant to predicting human health outcomes.

Reproductive and Developmental Toxicity

NICNAS4 reported a developmental and maternal toxicity NOAEL of 50 mg/kg/day for no specific species,

but the basis for that NOAEL was unclear.

Fort et al.30 examined the effect of triclosan, at larval exposure levels up to 32.3±9.43 µg/ml (measured 21-

day mean±SEM), on frog metamorphosis. A small marginally-significant acceleration in premetamorphic

development was reported, but the effect was not thyroid-mediated. Overall there was no effect on

metamorphosis. The authors suggested that the effect that was seen would be consistent with the reduced

bacterial stressors that would be found in the 50 L tanks used for the study.

Rodricks et al. (2010) summarized findings on developmental toxicity studies using mice, rats, hamsters,

and rabbits. Only in rats and mice were significant findings reported.

In a two-generation reproductive and developmental study using CRL:CD (SD)Br rats given triclosan at

doses of 0, 300, 1000, and 3000 ppm, body weights were significantly decreased in F1 animals on postnatal

days 14 and 21 in the high-dose group compared to controls. The viability index for high-dose F1 animals

was reduced, but the difference was not significant.

The SCCP (European Commission 2009) reviewed this same study and noted an absence of reproductivetoxicity at the 3000 ppm dose (~200 mg/kg/day for both sexes combined), but concluded that the NOAEL

for developmental effects would be 65 mg/kg/d (both sexes combined) because of pup body weight

decreases at the high dose.

Rodricks et al. (2010) reviewed a study in which CD-1 mice were given 0, 10, 25, 75, or 350 mg/kg/day on

gestation days 6 – 15, fetal body weights were significantly reduced in the two highest dose groups. The




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incidence/litter of irregular skull ossification was significantly increased in high-dose litters and the litter

averages for ossified forepaw and hind paw phalanges per fetus, possibly linked to developmental delay

due to the reduced fetal weights.

The SCCP (European Commission 2009) stated that taking maternal toxicity and fetal toxicity both into

consideration, there is no evidence of triclosan developmental toxicity (teratogenicity).

Endocrine Disruption

In vitro studies

Ahn et al.31reported results from a series of receptor-based bioassay systems for triclosan. Three receptors

were stably transfected: aryl hydrocarbon receptor (AhR – activates gene expression in a ligand-dependent

manner); estrogen receptor (ER); and androgen receptor. In each case the reporter gene was firefly

luciferase. In addition, the ryanodine receptor type 1 (RyR1) assay for compounds with potential to alter

Ca2+ homeostasis was performed using primary cultures of skeletal myotubes from wild-type mice.

In the AhR assay, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was used as a positive control. With TCDD

activation set at 100%, triclosan at 10 µM of compound was found to induce at 40.6±6.1%, suggesting

triclosan agonist activity. With both TCDD and triclosan added at 10 µM, induction reached only

70.4±2.1%, suggesting triclosan antagonist activity. Overall, the authors suggested triclosan would be a partial agonist against AhR.

In the ER assay, triclosan exhibited no direct estrogenic activity alone, suggesting no agonist activity.

When combined with estradiol, a 103 excess of triclosan reduced estradiol activity to 50% and a 105 excess

of triclosan reduced estradiol activity to 20%, suggesting antagonist activity.

While the authors stated that triclosan was a potent antagonist in the AR assay, no data were provided.

Ryanodine binding to microsomes enriched in RhR1 was significantly increased by 1.2 µM triclosan,

suggesting to the authors that triclosan is a dysregulator of cell Ca2+ homeostasis.

Gee et al.32

reported the estrogenic and androgenic activity of Ttriclosan using MCF7 human breast cancercells in culture and other in vitro assays. In MCF7 breast cancer cells in vitro, estradiol binding remained

>90% at 104 molar excess of triclosan. Half of estradiol binding to estrogen receptors (ER) was displaced

at a 106 molar excess of triclosan. In the estrogen-triggered ERE-CAT reporter gene in MCF7 cells, a 105-

fold excess of triclosan over 17β-estradiol effectively inhibited activation of the reporter gene and inhibited

17β-estradiol-induced cell growth stimulation.

In a seeming contradiction, the authors suggested that there was a small but not statistically significant

increase in MCF7 cell growth in the presence of triclosan alone. In a follow-up assay in which the cultures

were maintained for 21 days (the assay is normally done over 8 days), a statistically significant increase in

cell growth (but still less than a was reported for triclosan at 10-6 M and at 4 x 10-6 M, but not at lower (2 x

10-7 , 6 x 10-7) or a higher concentration (8 x 10-6).

The authors also performed a competitive binding assay between triclosan and testosterone to rat

recombinant androgen receptor (AR) protein, with the result that triclosan, at a 103 molar excess over

testosterone, reduced testosterone binding by around half and the decrease was linear when binding was

plotted versus the log of the molar ratio. They also determined growth stimulation in the presence of

triclosan in S115+A mouse mammary tumor cells and T24 human breast cancer cells.




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At a testosterone concentration of 10-9 M, S115+A cells are stimulated to grow and undergo a doubling in

number. Triclosan at 2 x 10-5 M prevented that growth.

Activation of the LTR-CAT reporter gene in stably transfected S115+A cells and in transiently transfected

T24 human breast cancer cells also was studied. In S115+A cells, 10-9 M testosterone triggered activation.

Triclosan at molar ratios to testosterone of 1 and 10 had no effect, but at a molar excess of 100, reduced the

activation by 20%. A molar excess of triclosan of 104 reduced activation by 40%. At a testosterone

concentration of 10-8 M, triggered activation in T24 cells. At a 103 molar excess of triclosan, the activation

was reduced by 30% and by 75% at a molar excess of 104.

James et al. (2009) postulated that triclosan is structurally related to inhibitors of estrogen sulfotransferase,

such as polychlorobiphenylols. To test potential enzyme inhibition, the authors harvested placental tissue

from almost term fetal sheep, homogenized the tissue and incubated the cellular material with triclosan for

~15 min. Estrone and 17-beta-estradiol were substrates and the effect of 4-hydroxy-3,3',4',5-

tetrachlorobiphenyl and 2'-hydroxytriclocarban on estradiol sulfonation was used for comparison. Triclosan

was a very potent inhibitor of both estradiol and estrone sulfonation suggesting competitive binding of

triclosan for estradiol sites on the sulfotransferase enzyme. The authors suggested that the effect of

triclosan as an inhibitor of estrogen sulfotransferase activity raised concern about the possible effects of

triclosan on the ability of the placenta to supply estrogen to the fetus, and in turn on fetal growth and

development.

Environ International Corporation (2010), in its analysis, reasoned that, were this phenomenon to be of any

significance, then administration of triclosan in vivo should have an impact on successful pregnancies. In a

two-generation rat reproductive and developmental toxicity study of triclosan at doses up to 3000 ppm

(described earlier), there was no evidence of an impact on reproductive performance, nor were there any

data to demonstrate that the ability to carry fetuses to term was compromised.

In vivo studies

Kumar et al.33 reported a clear no-effect level of 5 mg/kg/day (or higher) triclosan in a 60-day study of male

rats treated daily. Endpoints studied included decreased body weights (no-effect level of 20 mg/kg/day);

decreased testis, prostate, seminal vesicle, vas deferens and cauda epididymis weights (5 mg/kg/day);

down-regulation in the testicular levels of mRNA for cytochrome P450SCC, cytochrome P450C17, 3β-

HSD, 17-βHSD, StAR and AR as compared to control (10 mg/kg/day); decreased testicular 3β-HSD and

17β-HSD levels in vitro (10 mg/kg/day); and decreased serum hormone levels (10 mg/kg/day).

Zorilla et al.34 exposed weanling rats to 0, 3, 30, 100, 200, or 300 mg/kg/day of triclosan by oral gavage

from postnatal day (PND) 23 to 53. Predicated on the idea that the separation of the foreskin of the penis

from the glans penis, so-called preputial separation (PPS), is an early reliable marker of the progression of

puberty in the male rat, this gross endpoint was examined beginning on PND 33. Triclosan did not affect

growth or the onset of PPS. Serum testosterone and triiodothyronine (T3) were not different in a dose-effect manner. Total serum thyroxine (T4) decreased in a dose-dependent manner at 30 mg/kg and higher.

Thyroid stimulating hormone was not statistically different at any dose. Liver weights were significantly

increased at 100 mg/kg triclosan and above, but not in a dose-effect manner and other tissue weights were

not different from controls, exemplifying the difficulty in identifying a cohesive body of work on

Triclosan’s potential as an endocrine disruptor (or as an endocrine toxicant).




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In a follow-up study by Paul et al.,35 examined the question of how triclosan decreased serum T4 in vivo in

rats. The hypothesis was that triclosan upregulates rat hepatic catabolism and alters expression of cellular

transport proteins. The authors measured total serum T4, T3, and thyroid stimulating hormone (TSH).

Cytochrome P450 isoforms (Cyp2b1/2 and Cyp3a1/23) were determined enzymatically and as mRNA

expression levels using quantitative reverse transcriptase. Uridine diphosphate glucuronyltransferase

(UGT) activity, mRNA expression of UGT isoforms and sulfotansferase (SULT) isoforms, and mRNA

expression of hepatic transporters, including Oatp1a1, Patp1a4, Mrp2, and Mdr1, were also measured. All

mRNA expression assays were performed using reagent kits.

T4 levels decreased as expected as a function of triclosan concentration, with decreases at 100, 300, and

1000 mg/kg/day producing significant decreases, but not at 10 and 30 mg/kg/day. T3 levels were

decreased at 300 and 1000 mg/kg/day, but not at the three lower exposure levels. No significant

differences in TSH were found at any exposure, but the authors suggested that this may relate to T3

glucuronidation.

Cyp2b1/2 (at triclosan levels of 300 mg/kg/day) and Cyp3a1/23 (at Triclosan levels of 100 and 300

mg/kg/day) gene expression were increased, No significant effect was seen at lower doses. Liver

microsomal UGT activity was increased significantly only at 1000 mg/kg/day triclosan. UGT gene

expression was not significantly increased for Ugt1a6 or Ugt2b5 genes, but was increased at 100 and 300

mg/kg/day for Ugt1a1 genes.

SULT isoform expression was not dose-related for Sult1b1 (significantly reduced at 10 and 30 mg/kg/day,

but not at 100 or 300 mg/kg/day triclosan), but Sult1c1 expression was increased significantly at 100 and

300 mg/kg/day only.

No statistically significant changes were reported for mRNA expression of hepatic transporters.

The authors cautioned that, while these findings support a role for hepatic catabolism of T4 in the rat as a

likely mechanism of observed triclosan-induced hypothyroxinemia in the rat, the relevance to humans is

not established.

Allmyr et al. (2009) investigated if an everyday exposure to triclosan via triclosan-containing toothpaste for

14 days in 12 adult humans caused an increase in plasma 4β-hydroxycholesterol, indicative of CYP3A4

induction, and/or alterations in thyroid hormonal status. Plasma triclosan concentrations increased from

0.009–0.81 ng/g to 26–296 ng/g. The authors noted that the 296 ng/g plasma triclosan level is in the range

of triclosan plasma levels that could be attained with an oral dose of 4 mg triclosan. No significant changes

in plasma levels of either plasma 4β-hydroxycholesterol or thyroid hormones were reported during the

exposure. The authors concluded that triclosan-containing toothpaste use was not likely to alter metabolism

of drugs via CYP3A4 induction or cause adverse events because of thyroid disturbances in humans.

Other studies suggested weak estrogenic and androgenic effects, and antiestrogenic and antiandrogenic

effects (reported in fish/frogs or in vitro). Summary statements from Triclosan reviews already available

from various governmental sources on the import of those data are presented in Table 9.

The SCCP (European Commission 2009) did comment that data from a study using Japanese medaka fry

exposed to concentrations of triclosan up to 100 g/l for 14 days showed no effect on sex ratios in the

developing fish.




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Clinical studies

FDA6 summarized several studies reviewed by DeSalva et al.29 and by Lyman & Furia.36 None of the

studies indicated that triclosan at concentrations of < 25% causes sensitization, or that triclosan at

concentrations at

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< 0.5% causes irritation. In contrast, NICNAS4 stated that it had reviewed several studies

that had shown evidence of skin irritation, although applicable doses or references were not identified.

NICNAS4 stated that there is very limited evidence for triclosan causing photosensitization in healthy

volunteers or those with dermatological conditions. NICNAS4 also reported that humans orally

administered triclosan at

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< 30 mg/day for 15 or 42 days showed no evidence of any treatment effect.8

VI. Antibacterial/antimicrobial resistance9

FDA6 described two triclosan mechanisms of action for the inhibition of bacterial growth: 1) intercalation

into bacterial cell membranes and disruption of membrane activities (without causing leakage of

intracellular components, and 2) inhibition of bacterial type II fatty acid synthase enoyl-reductase (FabI

gene). Triclosan is bacteriostatic at low doses and bactericidal at high doses.

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In a 2002 report of the Scientific Steering Committee (SSC) of the European Commission Health &

Consumer Protection Directorate-General,37 the conclusion was reached that, at high (biocidal)

concentrations, triclosan is very effective and unlikely to produce a major problem of anti-microbial

resistance (e.g., all the microorganisms are dead). However, at sub-biocidal and bacteriostatic,

concentrations, triclosan is capable of penetrating bacteria and initiating changes related to important

mechanisms of antimicrobial resistance including possibly transferable mechanisms of resistance, though

the scientific evidence for transferability has been disputed. Sound scientific laboratory evidence exists for

the development of triclosan related mechanisms for antimicrobial resistance, but the evidence as to

whether these mechanisms are shared by other antimicrobial agents or whether they are transferable to

micro-organisms other than those used in the laboratory is limited and contradictory. Overall the SSC

noted that no evidence of such resistance has been seen in clinical isolates, and there is no epidemiological

evidence to suggest a problem in clinical practice.

A study by Cole et al.43 found no relationship between the use of triclosan and other biocides and antibiotic

resistance.

Lambert38 analyzed minimum inhibitory concentrations (MICs) from clinical strains of S. aureus (both

methicillin resistant (MRSA) and sensitive strains (MSSA)) and P. aeruginosa for changes from 1989 to

2000. While MRSA strains developed biocide resistance, MRSA antibiotic resistance has remained the

same. The same was true for MSSA strains. Overall this suggested to the author that any acquisition of

biocide resistance does not alter antibiotic resistance. And for P. aeruginosa, the MICs for triclosan were

actually reduced in 2000 compared to 1989, although the difference was not statistically significant.

The information that is available from studies of manufacturing sites

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and clinical follow up studies ofdental plaque flora which have failed to show biologically significant changes in MIC values to commonly

used antibiotics in patients using triclosan long term40,41 points to resistance patterns being stable over

periods of three to ten years.

Aiello et al.42 reviewed triclosan efficacy data along with data her laboratory developed on antimicrobial

resistance in use situations. Two studies reported findings from a randomized and masked intervention trial




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of 238 households using either 0.2% triclosan–containing liquid hand soap or plain soap over one year.

Neither of these studies demonstrated the emergence of antibiotic resistance associated with use of

triclosan-containing liquid hand soap, compared with plain soap.

Beier et al. (2008) characterized 50 vancomycin-resistant Enterococcus faecium (VRE) isolates from

human wastewater effluents in Texas. These VRE isolates were also resistant to 8 fluoroquinolone

antibiotics and most of the National Antimicrobial Resistance Monitoring System gram positive antibiotics.

The VRE isolates were sensitive to quinupristin/dalfopristin and to linezolid antibiotics and they were

sensitive to triclosan and other biocides. No cross-resistance or co-resistance between antibiotic resistance

and biocide susceptibility was found.

This contrasts with the work of Chen et al. who studied triclosan susceptibility to 732 pathogenic

Acinetobacter baumanii clinical isolates from hospitals in China. MIC values for triclosan ranged between

0.015 and 16 mg/l. They noted that these MIC values were lower than the in-use concentrations of

triclosan of 2000 to 20000 mg/l. They identified 20 (out of the 732) isolates for which the MIC was greater

than 1 mg/l and declared those to have reduced susceptibility to triclosan.

They then further examined those 20 isolates for antibiotic resistance and compared the results with 20

isolates with triclosan MIC values of 0.5 mg/l down to 0.03 mg/l. All 20 of the isolates with reduced

susceptibility to triclosan were resistant to amikacin, tetracycline, levofloxacin, and imipenem. Among the

20 isolates with triclosan MIC values < 0.5 mg/l, 11 were resistant to amikacin and tetracycline, and 8 were

resistant to levofloxacin and imipenem.

These authors further examined the potential that mechanism for triclosan reduced susceptibility, including

efflux pump over expression (in concept, if triclosan is removed from the bacterial cell, it is no longer

available to function as a biocide), but were unable to correlate expression of efflux pump genes with

triclosan reduced susceptibility. They identified mutations in the FabI (NADH dependent, enoyl-[acyl-

carrier-protein] reductase) gene in all 12 of the isolates with triclosan MIC values >4 mg/l and postulated

that triclosan resistance was linked to mutations in that gene.

Stickler and Jones (2008) studied clinical isolates of Proteus mirabilis in vitro to determine if exposure to

triclosan could result in decreased sensitivity (higher MIC values) to triclosan itself and/or increased

antibiotic resistance. Five strains of Proteus mirabilis were exposed in culture to triclosan at

concentrations from 0.5 to 10 mg/l for 5 days. Viable colonies (mutated to reduced triclosan susceptibility)

were subcultured and tested to determine triclosan MIC values and MIC values for trimethoprim,

ampicillin, ciprofloxacin, nitrofurantoin, norfloxacin, cephalexin, nalidixic acid, and gentamicin. The

parental strains and 2-3 mutant strains for each were tested. While mutant isolates were found with MIC

values for triclosan up to 60 mg/l, none of the mutated strains showed resistance to any antibiotic that was

different from the parent strain.

In 2009, SCENIHR 3

stated that triclosan at low concentrations acts by both inhibition of enoyl acylreductase mechanism, inhibition of energy-dependent uptake of amino acids, and possibly discharge of

membrane potential (as demonstrated in E. faecalis). The SCENIHR report concluded that current scientific

evidence (including bacteriological, biochemical and genetic data) does indicate that the use of certain

active substances in biocidal products in various settings may contribute to the increased occurrence of

antibiotic resistant bacteria. Some mechanisms of resistance are common to both biocides and antibiotics




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(e.g. efflux pumps, permeability changes and biofilms). The selective pressure exerted by biocides may

favor the expression and dissemination of these mechanisms of resistance.

Most recently, the European Commission’s Scientific Committee on Consumer Safety (SCCS) issued an

opinion on triclosan antimicrobial resistance (European Commission 2010) with the conclusion that the

available data have failed to demonstrate an increase in antibiotic resistance following triclosan use in situ.

Because in vitro studies have demonstrated that resistance to triclosan in bacteria is possible (see, for

example, Stickler and Jones (2008) above) and that there are mechanisms in bacterial resistance that can

result in cross-resistance to biocides and antibiotics (see for example, Chen et al. (2009) above), the opinion

went on note that it was not possible to draw an overall conclusion on whether the continuous use of

triclosan is involved in the development of resistance. The SCCS recommended prudent use of triclosan,

for example, in applications where a health benefit can be demonstrated and that additional research, for

example, on mechanisms of resistance, transfer of resistance, and translational studies from in vitro to in

situ situations.

VII. Exposure Assessment and Margins-of-Safety14

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A determination of triclosan exposures and margins-of-safety presumes that a hazard has been identified.

Rodricks et al. (2010) suggested that the statistically significant increases in nephropathy and stomach

pathology seen in male and female hamsters and the statistically significant effects in epididymides and

testes in male hamsters, all at the high-dose level of 250 mg/kg/day but not at 75 mg/kg/day could establish

a no-observable-adverse-effect-level (NOAEL) for repeated dose systemic toxicity. Further, in CD-1 mice

given 350 mg/kg/day on gestation days 6 – 15, fetal body weights were significantly reduced in the two

highest dose groups. The incidence/litter of irregular skull ossification was significantly increased in high-

dose litters and the litter averages for ossified forepaw and hind paw phalanges per fetus. None of these

effects were seen at 75 mg/kg/day, a presumptive NOAEL for developmental toxicity.

In addition, Rodricks et al. (2010) modeled the high-dose levels at which significant effects were seen

using the U.S. EPA’s benchmark dose approach. The lowest benchmark dose (BMDL) that provided the

best fit to the available male hamster nephropathy data was 46.91 (~47) mg/kg/day. All other hamster

endpoints for which there were statistically significant effects yielded BMDLs higher than that. The BMDL

that provided the best fit to the rat developmental toxicity data (body weight decreases in F1 animals) was

75.65 (~76) mg/kg/day. Noting that these two BMDLs were not inconsistent, the BMDL of 47 mg/kg/day

was recommended.

The SCCP (European Commission 2009) relied solely on a NOAEL value of 12 mg/kg/d based on

hematotoxicity in a chronic exposure study using rats. The SCCP noted that the mean plasma level of

28,160 ng/ml (± 12,928) from the 12 mg/kg/d dose group could be compared to human plasma levels, were

they available.

Exposure Assessment

Rodricks et al. (2010) noted that, for products that may be ingested, the daily triclosan intake is determined

by the amount of product used per day, the percentage of triclosan in the product, the amount of triclosan

absorbed by the GI tract, and the body weight of the subject. The corresponding calculation for use of

dermal products varies only in replacing GI tract absorption with dermal absorption. Combined oral and




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dermal intake for adult males and females were determined. Intake for children was also determined, but

utilized scaling to convert use rates for liquid body washes and body lotions from available adult usage.

For toothpastes, the adult male intake was 0.005 mg/kg/day; adult female, 0.006 mg/kg/day; and children,

0.023 mg/kg/day. For mouthwashes, the adult male intake was 0.003 mg/kg/day; adult female, 0.004

mg/kg/day; and children, considered to be zero.

For rinse off products such as liquid hand soap, liquid body washes, dish detergents, the adult male intake

was 0.007 mg/kg/day, combined; adult female, 0.009 mg/kg/day, combined; children, 0.011 mg/kg/day,combined.

For leave-on products such as body lotions, moisturizers, and deodorants, the adult male intake was 0.033

mg/kg/day, combined; female, 0.046 mg/kg/day, combined; children, 0.042 mg/kg/day.

Were all the products to be used on a daily basis, the intake estimates for adult males, adult females, and

children, respectively, would be 0.047, 0.064, and 0.074 mg/kg/day. For comparison purposes, data from

biomonitoring levels were converted from urine concentrations to intake estimates. For the 95th percentile

level (largest urine concentration levels reported), the intake estimates for adult males, adult females, and

children, respectively were 0.009, 0.007, and 0.004 mg/kg/day (5 – 20 times less than the product usage

based estimates).

The SCCP (European Commission 2009) determined systemic doses for oral products assuming 100%

availability of whatever triclosan was present, toothpaste use levels of 2,750 mg/d, mouthwash use levels of

30,000 mg/d, and triclosan content of 0.3% for toothpaste and 0.3% or 0.2% for mouthwashes. The

resulting systemic dose for toothpaste was 0.0234 mg/kg/d and for mouthwash was either 0.10 or 0.15

mg/kg/d for mouthwashes.

For leave-on cosmetics, the SCCP determined systemic doses using dermal absorption based on in vitro

data, triclosan content (ranged from 0.15 to 0.3%), surface area exposed (e.g., deodorant stick = 200 cm2

and body lotion = 15670 cm2), and one application per day. The resulting systemic dose for deodorant

stick was 0.0015 mg/kg/d, for body lotion at 0.15% and 0.3% triclosan was 0.0823 and 0.1646 mg/kg/d,

respectively. Face powder systemic doses ranged from 0.004 to 0.006 mg/kg/d and blemish concealer

doses ranged from 0.0003 to 0.0006 mg/kg/d.

For rinse-off cosmetics, the SCCP determined systemic doses using dermal absorption based on in vitro

data, a 10x dilution of triclosan in the product in use situations, and an 860 cm2 exposure area for hand soap

or 17500 cm2 for shower gel/body soap. The resulting systemic dose for hand soap was 0.0066 mg/kg/d

and, for shower gel/body soap, was 0.0268 mg/kg/d.

Table 10 presents a list of the systemic doses determined by the SCCP as a function of product type.

Margin of Safety

Rodricks et al. (2010) determined a margin of safety (MOS) by dividing the BMDL by the daily intake

estimate. For such a determination to be the most conservative, the daily intake should be the largest value

supportable by the available data and the BMDL should be the lowest value supportable by the available

data. For example, the MOS for body lotion usage for adult males is l808, for adult females is 1237, and

for children is 1119. These were the lowest MOSs reported. Were the biomonitoring levels (95 percentile)




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used instead, the MOS for body lotion usage for adult males would be 5222, for adult females would be

6714, and for children would be 11750.

Consideration was also given to the use of multiple products on a daily basis. Were each of the oral care

products, rinse-off products, and leave-on products applied on a daily basis, and were each of them

preserved with triclosan (unlikely, given the data in Table 5, where only 2% of baby shampoos contain

triclosan), then the MOS for adult males would be 1000, for adult females would be 732, and for children

would be 634. Rodricks et al. (2010) concluded that exposure to triclosan in consumer products is not

expected to result in adverse health effects in children or adults who use these products as intended.

The SCCP (European Commission 2009) used the rat NOAEL (which SCCP determined to be 12 mg/kg/d)

divided by the systemic dose delivered by products containing triclosan as given in Table 9 to determine an

MOS. For toothpaste, the MOS was 513 and for combined use of toothpaste, deodorant sticks, and hand

soap, the MOS was 381. For all products usage, which includes body lotion, the MOS values ranged from

49 to 32. The SCCP concluded that use of triclosan up to a maximum concentration of 0.3% in toothpastes,

hand soaps, shower gels/body soaps, and deodorant sticks is safe, any additional use in face powders and

blemish concealers at concentrations up to 0.3% also is considered safe, but use in other leave-on products

(e.g., body lotions) and in mouthwashes is not considered safe for consumer use.

Table 11 provides a side-by-side comparison of MOS determinations by Rodricks et al. (2010) and theSCCP (European Commission 2009).

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Triclosan is a chlorinated aromatic compound with functional groups representative of both phenols and

ethers. Its IUPAC name is 5-Chloro-2-(2,4-dichlorophenoxy)phenol. Triclosan may function in cosmetic

formulations as a cosmetic biocide, deodorant agent, or preservative. At ambient temperatures, triclosan is

a crystalline powder, so any material supplied as triclosan in a liquid form, must, by definition, be a

mixture with a solvent. Triclosan is supplied to cosmetic formulators under several trade names and in

several trade name mixtures.

Information on the use concentration of triclosan in cosmetics as a function of cosmetic product type is

available from the VCRP maintained by the FDA, but this information is likely underreported. Use

concentration data as a function of product type is limited (not all reported uses have use concentrations),

but use concentrations in cosmetics appear to be in the 0.01 - 0.3% range. Triclosan also is used in some

product categories that raise the possibility of user exposure to aerosols. Most aerosol particles from

cosmetic products, however, are sufficiently large such that they are deposited in the nasopharyngeal region

and are not respirable.

Analysis of triclosan imported from India and China uncovered the presence of dioxin and furan impurities.

USP and the government of Canada have established limits for such impurities.

Independent of the presence of dioxin impurities in triclosan as supplied to cosmetics formulators, there is a

question regarding the possibility that triclosan in cosmetic formulations applied to the skin may

photodegrade to dioxin compounds on exposure to light. Triclosan can photodegrade to 2,7- and 2,8-

dichlorodibenzo- p-dioxin, and 2,4-dichlorophenol, but the effect is pH dependent. The relevance of these

photodegradation products to the safety of triclosan in cosmetics is not established.








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Triclosan is slowly and not extensively absorbed by the dermal route, but is rapidly and well absorbed by

the oral route. Triclosan measured in rodent radioactivity studies (following oral and dermal exposures)

indicate distribution to the liver, lung, kidney, gastrointestinal tract, and gall bladder. Triclosan absorbed

from the gastrointestinal tract undergoes extensive first-pass metabolism, which primarily involves

glucuronide and sulfate conjugation. Triclosan is also metabolized to its glucuronide and sulfate conjugates

by the skin. Triclosan glucuronide is predominantly excreted in the urine, and triclosan is predominantly

excreted in the feces. Triclosan administered orally and dermally is excreted in greater concentrations in

the urine than in the feces in humans.

Triclosan has low acute toxicity by all evaluated routes in all evaluated species.

Repeat dose toxicity has been evaluated in the baboon (oral route), rat (oral, and inhalation routes), mouse

(dermal route), rabbit (dermal), and hamster (oral). Triclosan NOAELs based on local irritation effects

tend to be < 10 mg/kg/day by all routes, except inhalation, which has a reported NOAEL of 5 x 10-5 mg/m3.

LOAELs and NOAELs based on systemic toxicity tend to be <1000 mg/kg/day, with no obvious common

toxicity among studies and species. Statistically significant increases in nephropathy and stomach

pathology were reported in male and female hamsters and statistically significant effects were reported in

epididymides and testes in male hamsters, all at the high-dose level of 250 mg/kg/day but not at 75

mg/kg/day.

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Triclosan does not appear to have significant reproductive/fertility/developmental toxicity. Triclosan has

been linked to hypothyroxinemia in rats and has been suggested as having potential to disrupt the thyroid

axis in amphibians. In rats, hypothyroxinemia via a hepatic catabolism mechanism has been suggested, but

the implications for human exposure is unclear. One recent study in frogs reported a marginal acceleration

of pre-metamorphic development by a non-thyroid mechanism in amphibians, with no overall alteration in

metamorphosis. In CD-1 mice given 350 mg/kg/day on gestation days 6 – 15, fetal body weights were

significantly reduced in the two highest dose groups. The incidence/litter of irregular skull ossification was

significantly increased in high-dose litters and the litter averages for ossified forepaw and hind paw

phalanges per fetus. None of these effects were seen at 75 mg/kg/day, a presumptive NOAEL for

developmental toxicity.

In various assays for endocrine disruption effects, triclosan gave weak responses, although one study did

report competitive binding to the estrogen receptor sufficient to support growth of an estrogen-dependent

cell line and another study reported binding to the thyroid hormone receptor.

Trisclosan has been evaluated in a number of standard (and other) genotoxicity assays, including bacterial

reverse mutation tests, in vitro mammalian cell gene mutation test, and in vitro mammalian chromosome

aberration tests, a mammalian bone marrow chromosomal aberration test, and an unscheduled DNA

synthesis assay in mammalian cells in culture --- except in one (an in vitro cytogenetic assay with Chinese

hamster lung fibroblasts), the findings were negative. Based on the weight of evidence, triclosan is not

genotoxic.

Rat, mouse, and hamster carcinogenicity studies are available and have been reviewed extensively with

mixed interpretations. Rat and hamster oral chronic toxicity/carcinogenicity studies found no carcinogenic

potential for triclosan in rats at < 3000 ppm and in hamsters at < 250 mg/kg/day. However, a mouse oral

chronic/carcinogenicity bioassay was positive for carcinogenicity based on an increased incidence of liver

neoplasms in male and female mice at

40

41

>30 mg/kg/day. Presuming that activation of peroxisome42




25

1

2

3

4

5

proliferator activated receptor alpha is the primary mode of action for triclosan-induced

hepatocarcinogenesis in mice, these findings did not support either a mutagenic mode or cytotoxic mode of

action. FDA has nominated triclosan for dermal carcinogenicity study under the NTP.

In rabbits, triclosan is a moderate dermal irritant as well as a moderate eye irritant, but when tested at

concentrations < 0.5% in various formulations did not cause dermal toxicity. Triclosan is, at most, a weak

sensitizer, but when tested at concentrations of

6

< 25% in formulation, no sensitization was reported.

Triclosan is not a phototoxicant in guinea pigs.

7

8

9

10

11

12

13

14

15

16

17

1819

20

21

22

23

24

25

26

27

Triclosan is bacteriostatic at low concentrations and bactericidal at high concentrations. At high (biocidal)

concentrations, Triclosan is very effective and unlikely to produce a major problem of anti-microbial

resistance (e.g., all the microorganisms are dead). However, at sub-biocidal and bacteriostatic,

concentrations, triclosan is capable of penetrating bacteria and initiating changes related to important

mechanisms of antimicrobial resistance including possibly transferable mechanisms of resistance. In actual

usage, however, no evidence of such resistance has been seen so far in clinical isolates, and there is no

epidemiological evidence to suggest a problem in clinical practice. Although, the stability and persistence

of triclosan biocidal resistance has not been widely studied, the limited information available points to

resistance being stable over a three to ten year period. One study found no relationship between the use of

triclosan and other biocides

and antibiotic resistance in homes where biocidal products were

or were not being used.

Different approaches have been described for determining the systemic dose that would result from use of

triclosan-containing products, although the maximum use concentration of triclosan in those products is

given by 0.3% by all. Resulting systemic doses from use of triclosan-containing products have been

compared to the dose determined in animal studies to be a NOAEL, but the NOAEL value chosen in one

case was 12 mg/kg/d based on hematotoxicity in rats and in another case was 47 mg/kg/d based on

nephropathy in hamsters. Resulting MOS values have ranged from a low of 32 for use of all products by

one analysis to a high of 47000 for hand soap use by another analysis.




26

O

OHCl

Cl

Cl

12

34

56

7

89

10

11

12

13

14

15

16

17

1819

20

2122

O

OH

Cl Cl

Cl

Figure 1. Triclosan (5-Chloro-2-(2,4-dichlorophenoxy)phenol (IUPAC name))

O

OCl

Cl

Cl

Triclosan High pH + heat Trichlorodibenzo-p-dioxin

Figure 2. Conversion of triclosan to trichlorodibenzo-p-dioxin

(for illustrative purposes, the structure of triclosan is pictured in Figure 2 with the phenol moiety

rotated around the –O bond so that the proximity of the hydroxyl group and conversion to a dioxincompound can be more readily seen).

O

O-

Cl

Cl

Cl 23

24

25 Figure 3. Phenolate form of Triclosan at pH > 8.1.




27

1 Table 1. Regulatory Decisions on Triclosan.

Authority Product Name or

Category

Concentration Limit/Restriction Comment

U.S. (FDA)44-47

Drugs Total Toothpaste (Colgate-

Palmolive Co.)

0.30% OTC dentifrice to treat gingivitis44

Acne therapeutic Proposed: leave-on: 0.2-0.5%rinse-off:0.3-1.0%

Under review as an OTC45

Soap and deodorant Antibacterial soaps generally contain <

0.3% triclosan.45

Antibacterial claim

Devices TempBond Clear with

Triclosan (Sybron Dental

Specialties, Inc.)

NR Regulated as a temporary dental cement. 46

MONOCRYL* Plus

(Poliglecaprone 25)

Antibacterial Suture

(Ethicon Inc.)

< 2360 µg/m Regulated as an absorbable surgical suture47

Europe48 Cosmetic products < 0.3%

Canada49 Cosmetic products

(mouthwashes excluded)

< 0.3% in other cosmetic products

All oral products < 0.03% in mouthwashes not to be used by children < 12 years of age

and labeled “do not swallow”

polychlorinated dibenzo-p-dioxin (PCDD)and polychlorinated dibenzofuran (PCDF)

impurities <0.1 ng/g 2,3,7,8-tetra-

chlorodibenzo-p-dioxin and 2,3,7,8-tetra-

chlorodibenzofuran; and <10 µg/g total

other PCDD/PCDF impurities, with noindividual impurity greater than 5 µg/g

Japan 50 Cosmetic products as a preservative

<0.10 g/100 g product (<0.1%)

Australia4 Cosmetic products < 0.3% Provisional recommendation. Eye, skin,

respiratory system irritant.

Recommendation for compliance with USP

limits for dioxins and dibenzofurans

(synthesis impurities).

Norway51 Cosmetic products “… should be restricted.” Concern about bacterial resistance to

triclosan and to clinically important

antimicrobial agents.

NR = not reported

2

3




28

1 Table 2: Triclosan Physicochemical Properties

Property Value and Conditions Reference

Molecular weight 289.54111

Physical state white crystalline powder4,13

Specific Gravity 1.55 X 103 kg/m3 11

Density 1.55 g/cm3 at 22° C4

Acid Dissociation Constant (pKa) 8.14 at 21° C11

pH Not available11

Stability

Neat triclosan is stable to UV radiation4

Triclosan solutions are not stable to chlorine4

Stable under normal storage conditions (ambient temperature)

when tested after 4 and after 9 years European Commission 2009

Melting point 56.5° C11

54 – 57.3° C13

Boiling point 280 – 290° C (decomposes)13

Water solubility

0.012 g/l at 20° C11

20 mg/l at 20° C13

Octanol-water partition coefficient (Log Po/w)

4.8 at 25° C 11

4.7613

Vapor Pressure

5.2 x 10-6 mm Hg at 25° C11

2.2 x 10-6 mm Hg at 20° C

4 x 10-6 mm Hg at 20° C13

2

3




29

1

2

Table 3. Measured 2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) and 2,3,7,8-Tetrachloro-

dibenzofuran (TCDF) impurities in Triclosan from India and China.52

Sample # Country TCDD (pg/g)a TCDF (pg/g) a

1 India 17.2 0.70

2 China 95.4 7.13

3 India 111.8 3.43

4 India 41.5 8.51

5 India 1712.0 0.43

6 India 18.9 207.3

3

a those values in excess of USP specifications

11are highlighted. 4




30

1 Table 4. USP and Health Canada’s Limits on Triclosan Impurities

Impurity USP 32 (2009)11 Canada49

2,4 Dichlorophenol < 10 µg/g (< 10 ppm or 0.001%) NA

3-Chlorophenol < 10 µg/g (< 10 ppm or 0.001%))

4-Chlorophenol < 10 µg/g (< 10 ppm or 0.001%)

2,3,7,8 Tetrachlorodibenzo-p-dioxin <1 pg/g (1 ppt)a < 0.1 ng/g

2,3,7,8 Tetrachlorodibenzo-furan <1 pg/g (1 ppt)a < 0.1 ng/g

2,8-Dichlordiobenzofuran < 0.25 µg/g (< 0.25 ppm or 0.000025%) < 10 µg/g total other PCDD/PCDF

impurities, with no individual impurity

greater than 5 µg/g2,8-Dichlorodibenzo-p-dioxin < 0.5 µg/g (< 0.5 ppm or 0.00005%)

1,3,7 Trichlorodibenzo-p-dioxin < 0.25 µg/g (< 0.25 ppm or 0.000025%)

2,4,8 Trichlorodibenz-furan < 0.5 µg/g (< 0.25 ppm or 0.000025%)

Other Contains not less than 97.0% triclosan

calculated on the anhydrous basis. Not more

than 0.5% of total impurities. Not more than

0.1% of any individual impurity.

manufacturers must possess: raw

material specifications for triclosan;

identification of analytical method used

to determine PCDD and PCDF levels;

and finished product specifications

NA = Not applicable or not specified

PCDD/PCDF = polychlorinated dibenzo-p-dioxin / polychlorinated dibenzofuran

a Calculated as follows: (1 pg/µl) x (10 µl) x (1/30 g) x (30 µl) = 1 pg/g = 1 ppt52

2

3




31

1 Table 5. Frequency of use and use concentrations of Triclosan in cosmetics.

Product Category Total number of products in

each product category53

Number of products containing

Triclosan in each product

category53

Concentration of Use (%) 20

Baby Products a

Shampoos 56 1 (~2%) None reported

Lotions, oils, powders, and creams 137 3 (~2%) None reported

Other 143 None reported None reported

Baby products subtotal 336 4 (~1%) None reported

Bath products

Oils, tablets, and salts 314 1 (<1%) None reported

Bubble baths 169 None reported None reported

Capsules 4 None reported None reported


Bath products subtotal 721 1 (<1%) None reported

Eye Makeup

Eyebrow pencils 144 None reported None reported

Eyeliners 754 None reported None reported

Eye shadow 1215 18 (~1.5%) 0.05%

Eye lotions 254 2 (~1%) None reported

Eye makeup remover 128 None reported None reported

Mascara 499 2 (<1%) None reported

Other 365 6 (~2%) None reported

Eye makeup subtotal 3359 28 (~1%) 0.05%

Fragrance products

Colognes and toilet waters 1377 27 (2%) 0.1%

Perfumes 666 None reported None reported

Powders 221 5 (~2%) None reported

Sachets 12 None reported None reported


Fragrance products subtotal 2842 42 (~2%) 0.1%

2




32

1





category53


Non-coloring hair care products

Conditioners 1226 1 (<1%) 0.05%

Sprays/aerosol fixatives 312 None reported None reported

Straighteners 178 None reported None reported

Permanent waves 69 None reported None reported

Rinses 33 None reported None reported

Shampoos 1361 None reported b 0.04 - 0.2%

Tonics, dressings, etc. 1205 1 (<1%) 0.1%

Wave sets 51 None reported None reported

Other 807 1 (<1%) None reported

Non-coloring hair care products

subtotal

5242 3 (<1%) 0.04 – 0.2%

Hair coloring product c

Dyes and colors 2393 None reported None reported

Tints 21 None reported None reported

Rinses 40 None reported None reported

Shampoos 40 None reported None reported

Color sprays 7 None reported None reported

Lighteners with color 21 None reported None reported

Bleaches 149 None reported None reported


Hair coloring products subtotal 2839 None reported None Reported

2




33

1





category53


Makeup

Blushers (all types) 434 1 (<1%) 0.2%

Face powders 661 None reported 0.2%

Foundations 589 5 (~1%) 0.1%

Leg and body paints 29 None reported None reported

Lipsticks d 1883 None reportedd None reported

Makeup bases 117 1 (1%) None reported

Rouges 102 None reported None reported

Makeup fixatives 45 None reported None reported

Other 485 3(<1%) 0.3%

Makeup subtotal 4345 10 (<1%) 0.1 – 0.3%

Nail care products

Basecoats and undercoats 79 None reported None reported

Cuticle softeners 27 None reported None reported

Creams and lotions 14 None reported None reported

Extenders 2 None reported None reported

Nail polishes and enamels 333 None reported None reported

Nail polish and enamel removers 24 None reported None reported


Nail care products subtotal 617 1 (<1%) None Reported

Oral hygiene products

Dentifrices 59 None reported None reported

Mouthwashes and breath

fresheners e

74 None reported e 0.04%


Oral hygiene products subtotal 219 None reported 0.04%

2




34

1

Product Category Total number of products

in each product category53



category53


Personal hygiene products

Bath soaps and detergents 1665 45 (~3%) None reported

Underarm deodorants 580 162 (28%) 0.2 - 0.3%

Douches 14 None reported None reported

Feminine deodorants 19 None reported None reported

Other 792 19 (~2%) 0.3%

Personal hygiene products

subtotal

3070 226 (~7%) 0.2 - 0.3%

Shaving Products

Aftershave lotions 367 2 (<1%) None reported

Beard softeners 3 None reported None reported

Mens talcum 3 None reported None reported

Preshave lotions 22 None reported None reported

Shaving cream 122 3 (2.5%) None reported

Shaving soap 10 None reported None reported

Other 134 6 (4.5%) None reported

Shaving products subtotal 661 11 (~2%) None Reported

Skin care products

Skin cleansing creams, lotions,

liquids, and pads

1446 31 (2%) 0.01-0.3%

Depilatories 42 Not reported None reported

Face and neck creams, lotions, etc. 1583 30 (2%) 0.1%

Body and hand creams, lotions, etc. 1744 27 (1.5%) 0.1%

Foot powders and sprays 47 6 (~13%) None reported

Moisturizers 2508 28 (1%) None reported

Night creams, lotions, powder and

sprays

353 14 (4%) None reported

Paste masks/mud packs 441 12 (~3%) None reported

Skin fresheners 259 3 (~1%) None reported





35

Skin care products subtotal 9731 162 (~2%) 0.01-0.3%

Product Category Total number of products

in each product category53



category53


Suntan products

Suntan gels, creams, liquids and

sprays

107 2 (~2%) None reported

Indoor tanning preparations 240 1 (<1%) None reported


Suntan products subtotal 409 3 (<1%) None reported

Total usage/concentrations-of-use

range across all product categories 34391 491 (~1.5%) 0.01-0.3%

a For baby shampoos reported, only ~2% contain Triclosan, or conversely, the vast majority do not, and the situation is similar for the baby lotions, powders, and creams category. While uses of Triclosan were reported to FDA under the VCRP for each of these categories, no use concentrations

were provided in the industry survey. And no uses of Triclosan or use concentrations were reported for the 143 products in the “other” baby

products category. b Triclosan is reported to be listed on the label of Oscar Blandi Pronto Dry Shampoo ( http://www.cosmeticsdatabase.com )c None of the 2839 hair coloring products were reported to contain Triclosan in the VCRP and no use concentrations were reported by industry.d Triclosan is reported to be listed on the label of Revlon Colorstay Overtime Lip Color Glossy Topcoat ( http://www.cosmeticsdatabase.com )e

While no reported uses were submitted to the VCRP, a use concentration was reported in the Council survey, so it must be presumed there is atleast one use.

1


http://www.cosmeticsdatabase.com/






36

1 Table 6. Triclosan in urine from NHANES subjects. Calafat et al. (2008).

Urine concentrations (μg/L)

Group Sample size Geometric mean 50th percentile 95th percentile

All 2514 13.0 9.2 459.0

6 - 11 years 314 8.2 5.9 148.0

12 - 19 years 713 14.5 10.2 649.0

20 - 59 years 950 14.7 10.3 491.0≥60 years 537 10.3 6.5 386.0

All male 1228 16.2 11.7 566.0

All female 1286 10.6 7.4 363.0

2




37

1 Table 7. Triclosan route-specific and species-specific repeat-dose NOAELs.4-7

Route, Duration, Species NOAELa Comments

Dermal

Subacute

Rat 3.0 mg/kg/day LOAEL = 6.0 mg/kg/day (local effects at application sites)

Rat 7.5 mg/kg/day, males

3.5 mg/kg/day, females

Basis for NOAEL selection: irritation

Mouse 0.6 mg/animal

(100 µg/cm2)

LOAEL = 1.5 mg/kg/day (dermal irritation and increased

absolute and relative liver weights)

Rabbit 15% None.

Subchronic

Rat 40 mg/kg NOAEL based on systemic toxicity, characterized as occult

blood in urine (EPA) or lack thereof (NICNAS). Each Agency

may have drawn a different conclusion from the data, which

NICNAS stated were unreliable evidence of systemic toxicity.

Rat 80 mg/kg

Rat 10 mg/kg/day NOAEL from above study, but based on local irritation –

reversible after a 20 day recovery period.

Rat 2.5%, 5% None

Inhalation

“All Durations”

Rat None assigned LOAEL = 3.21 mg/kg/day, males, 9.91 mg/kg/day females.

Based on increased total leucocyte count and serum alkaline

phosphatase.

Subacute

Rat NOAEC

(irritation): 5 x 10-5 mg/m3 b

LOAEL (systemic): 1300 mg/m3, clinical signs of toxicity and

death after 2 days of dosing

Oral

Subacute

Baboon 30 mg/kg None

Subchronic

Dog 12.5 mg/kg Effects observed at all doses; NOAEL based on reversal of

serum alkaline phosphatase elevations after 28-day recovery

period.

Rat 1000 ppm (52.4 mg/kg/day) LOAEL: 3000 ppm (168.0 mg/kg/day) based on liver histopath.

Mouse None assigned LOAEL: 25 mg/kg/day. Based on hematology parameters,

relative liver weights and total cholesterol. NICNAS excluded




38

Route, Duration, Species NOAELa Comments

this study from its risk assessment due to mechanistic

differences between humans and mice.

Chronic

Baboon 30 mg/kg Based on diarrhea at LOAEL of 100 mg/kg/day

Rat 40 mg/kg/day, males

56 mg/kg/day, females

NOAEL based on histopath. changes in male liver and a trend

for reduced body weight in females (LOAEL not reported).

Rat 1000 ppm (52.4 mg/kg/day) LOAEL of 3000 ppm (168.0 mg/kg/day) based on significant

body weight decreases (both sexes) and non-neoplastic liver

changes in males.

Mouse 10 mg/kg/day (systemic) Based on neoplasms (both sexes) at 30 mg/kg/day [interpreted

as mouse-specific and PPAR α-,mediated]

Hamster 75 mg/kg/day Based on LOAEL of 250 mg/kg/day (both sexes) based on

decreased body weight gains, mortality, nephropathy, and

histopathology (stomach and testes).

1

2

3 Table 8. Hepatic carcinomas and adenomas in mice as a function of triclosan dose.

Number of tumor bearing micea

Carcinoma Adenoma Combined

Dose

(mg/kg/day) Males Females Males Females Males Females

0 2 0 5 (5) 0 6 (6) 0

10 3 (3) 0 7 (7) 1 10 (10) 1

30 6 (3) 1 (1) 13 (16) b 3 (3) b 17 (17)c 3 (3) b

100 11 (9)c 1 (1) 22 (24) c 6 (6) c 32 (32) c 6 (6) c

200 24 (22) c 14 (14) c 26 (26) c 11 (11) c 42 (42) c 20 (20) c a results in parentheses are based on a pathology peer review conducted after the study using the

liver slides prepared during the study.

4

5

6

7

8

b statistically significant at p≤0.05.

c statistically significant at p≤0.01.




39

1

2

Table 9. Observations made in various governmental reviews regarding triclosan endocrine

disruption effects in fish, frog, and in vitro preparations.

Fish Weakly androgenic, weakly estrogenic, toxic (altered fin

length, sex ratio, etc.) 5

Preliminary data indicate that triclosan (or metabolite) is not

potently estrogenic to freshwater fish but it may be weakly

estrogen, anti-estrogen or androgenic.

4

Frog Induces estrogen antagonism following intraperiotoneal

injection of high doses; reduced testosterone levels at lower

doses. Binds to thyroid hormone receptor.5

In vitro Competively binds to estrogen receptor and supports growth

of this estrogen-dependent MCF-7 cell line cell line. Binds to

rat androgen receptor 5

3




40

1

2

Table 10. Systemic triclosan doses determined as a function of product type containing triclosan

(European Commission 2009).

Product Triclosan content

(%)

Systemic triclosan dose

(mg/kg/d)

Toothpaste 0.3 0.0234

Hand soap 0.3 0.0066

Shower gel/body soap 0.3 0.0268

Deodorant 0.3 0.0015

Mouthwash 0.2 0.1000

0.3 0.1500

Face powder 0.2 0.0040

0.3 0.0060

Body lotion 0.15 0.0823

0.3 0.1646

Blemish concealer 0.15 0.0003

0.3 0.0006

Toothpaste, hand soap, shower gel/body soap, deodorant

combined

0.3 0.0583

Mouthwash, body lotion, face powder, blemish concealer

combined

0.15 - 0.3 0.1866

0.3 0.3212

All products 0.15 - 0.3 0.2449

0.3 0.3795

3

4




41

1 Table 11. Comparison of margin of safety (MOS) determinations.

Product MOS from Rodricks et al. (2010) MOS from

SCCP (European

Commission

2009)

adult male adult female child

toothpaste 9400 7834 2043 513

mouthwash 15667 11190 ND 80 - 120

hand soap 47000 47000 9216 1118

body washes 11750 9400 7833 448

body lotion 1808 1237 1119 73 - 146

deodorant 15667 15667 ND 8000

combined exposures 1000 732 634 32 – 49

2

3

4

ND = not determined




42

1

23

4567

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http://www.ciba.com/ind-pc-triclosan-global-steward-ups.htm




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Data



Green Book 1 Tab: Data

Panel Book Page Data/Document Title or Description

51 Environ Endocrine Description Commentary

94 Rodricks et al

157 SCCP opinion on safety

283 SCCP opinion response to public comments

299 SCCS opinion on antimicrobial resistance

355 Council comments on SLR

359 Environ comments on SLR




r p v o d ôr

nvestigation of Potential n docrine tivity of Triclosan

1.

BACKGROUND

Recent studies have investigated the effect of triclosan on thyroid hormone levels

Crofton et al. 2007; Paul et al. 2009; Zorrilla et al. 2009 or other serum hormone levels,

e.g., testosterone Kumar et al. 2008, 2009; Zorrilla et a]. 2009 or estradiol James et al.

2009 , in studies conducted to assess the potential that triclosan could function as an

endocrine disruptor in mammalian species.

Triclosan has been tested in an extensive battery of tests to include studies with

human subjects on safety and tolerability and studies in experimental animals conductedover a wide range of doses and durations in several species to assess the potential for

general systemic toxicity, carcinogenicity, mutagenicity, neurotoxicity and reproductive

andlor developmental toxicity as reviewed by Rodricks et al. 2009 . None of these tests

reported effects that were specifically attributed to endocrine disruption activity;

however, the tests were not designed to evaluate subtle modulations in endocrine

function. Yet, these data along with recently published studies that did investigate the

potential for endocrine action by triclosan could be used, in an integrated manner, to

assess the following: if triclosan were acting by modulation of endocrine function and

targeting endocrine responsive organs, i .e ., the thyroid or reprodu tive system, then what

would be the expected signs of toxicity that should have been manifested in the studies

already conducted?

An important consideration in this investigation was the applicability of the data

reviewed to assess impacts on human endocrine system function. To address this, a

hierarchical approach was taken and value assigned in descending order to the following

types of studies: studies in human volunteers; 2 studies conducted in v ivo in

mammalian species; 3 studies conducted in vitro with primary cells or tissues harvested

from mammalian species; and. 4 in vitro studies in immortalized cells, in particular

immortalized cancer cell l ines whether human or animal cell lines. Use of this latter

category, immortalized cell lines, requires careful scrutiny. In order to use in vitro data to

be predictive of human health outcomes, extrapolation from an in vitro system to an in




Investigation of Potential Endocrine Activity of TrIclosan September 24, 2009

vivo model and in the case of non-primate cells, extrapolation across species e g animal

to human, must be considered. More importantly is consideration of the genomic

behavior of immortalized cells, in particular immortalized cancer cell lines, especially

when transcriptional and receptor activation may be involved. Gentry et al. 2009

reported that the genomic activation profile in response to xenobiotics may be

considerably different in immortalized and cancer cell l ines than in primary cells even at

comparable doses of the compound.

The approach taken in this investigation was to:

• eviewrecent in vivo and in vitro data for each endpoint that could beimpacted, e.g., the thyroid and reproductive system;

• Define what would be expected to be seen in terms of signs and symptoms iftriclosan were acting to disrupt endocrine function;

• Compare the expected outcome to the data that have been reported in thehuman safety and experimental studies to identif’ those reported effects thatcould be the consequence of endocrine disruption; and,

• Discuss the potential for biologically significant effects in humans followinglong-term, low-level exposure.

The investigation of the recent literature for triclosan identified two major systems that

could be impacted if triclosan was acting as an endocrine disruptor, i.e., the thyroid and

the reproductive system. This investigation focused on those two potential targets for

triclosan induced endocrine activity.

2.0 POTENTIAL EFFECTS ON THE THYROID

2.1 Recent Thyroid Hormone Studies

Three studies have been conducted in rats that evaluated the effects of triclosan on

total serum thyroid hormone concentrations Crofton et al. 2007; Paul et al. 2009; Zorrilla

et al. 2009 and a study in human volunteers has been conducted Alimyr et al. 2009 .

Studies in rats in which changes in thyroid hormone levels and alteration in thyroid

histopathology are evaluated serve as an initial screen of the potential for a xenobiotic to

alter thyroid function in humans DeVito et al. 1999 . However, changes in thyroid

hormone levels in rats do not necessarily mean that thyroid function in rats will be altered

2




Investigation of Potential ndocrine Activity of Triclosan September 24, 2 9

or that physiological systems in rats will be adversely affected. or do these changes in

serun-i thyroid hormone levels in rats translate quantitatively to similar changes in humans

because of the differences between rats and humans in both the capacity to keep thyroid

hormones within normal physiological ranges but also because of the decreased

sensitivity of humans to these changes. As discussed in the following sections, changes

in thyroid hormone levels in rats, in particular in short-term studies, in which initial

adaptive mechanisms may be operative, are reviewed in the context of all of the

experimental data for triclosan.

2.1.1 Review of studies in rats

The study by Crofton et a . (2007) was conducted to evaluate the effects of

triclosan on circulating levels of thyroxine T The protocol was designed based on

data for other chemicals in which a 4-day exposure to rats had previously been used to

assess the potential for an effect on thyroid hormone levels (Zhou et al. 2001; Craft et al.

2002; Crofton 2004). In the Paul et al. (2009) study, the effect of triclosan on levels in

dams and in offspring was investigated. The Zorrilla et al. (2009) study was designed to

investigate the effects of triclosan exposure during pubertal development on thyroid

hormone levels and sexual development in male rats.

In the Crofton et a . (2007) study, young female Long-Evans rats (8 to 16 rats pergroup) age 27 to 29 days were randomly assigned to treatment groups and administered

0, 10, 30, 100, 300, or 1000 mg triclosanlkg day by oral gavage in corn oil for 4

consecutive days. No clinical signs of toxicity were seen in any animals during treatment.

Twenty-four hours after the last dose, rats were sacrificed, trunk blood was collected and

body and liver weights were determined. No treatment-related effects on body weight

gains were found. Absolute and relative liver weights were increased in the 1000

mgilcg/day dose group bu t no t in any of the other treatment groups. No o th er organs were

weighed nor were histopathological examinations conducted.

After 4 days of treatment, dose-dependent decreases in total serum (bound and

free) concentrations were seen in rats treated with triclosan at concentrations of 100, 300.

or 1000 mg/kglday with decreases of 28 , 34 , and 53 , respectively. The study

authors d id not offer an opinion as to the consequences of decreases in circulating total




Investigation of Potential Endocrine Activity of Triclosan eptember 24 2 9

levels nor whether a reduc tion o 53 in circu lat ing tota l levels would affec t

phys iological func tion in these female rats. Serum concen tra tion s o tri iodothyronine

T the more bioactive thyroid hormone , or thyroid stimulating hormone TS H) were

not measured.

In the Paul et a]. 2009) study’ pregnan t Long Evans ra ts age was no t specified)

rece ived triclosan by gavage in corn oil at doses o 0 30, 100 or 300 mg/kg/day

beginning on gestationa l day GD) 6 and con tinu ing da ily until post-natal day PND) 21.

At PND 22, there was a significant decrease 3 0 ) in total serum concentrat ion in

dams given 300 mg/kg /day triclosan bu t other doses were not sign ificantly different from

control values number o animals p r dose group was not given). A similar pe rcentage

reduction in total serum

concentrations o approximately 30 was seen in offspr ing o

dams in the 300 mg/kg/day dose group when measured at PND 4; how ever, no effects on

total serum concentra tions were seen in these offspring when levels were measured

at PND 14 or 212 and no effects on se rum leve ls we re seen in offspring in the two

low er dose groups at any time po int measured postnatally. The similarity in patte rn o

total concentrations in offspr ing at PND 4 with maternal leve ls at PND 22

suggested, according to the authors, that the dam supp lied thyroid hormones to the fetus.

While maternal serum concentra tions were not measured on PND 4 given th e rapid

metabo lism and excretion o triclosan from the body R odr icks et al. 2009) , steady state

levels, and hence effec ts on levels, may have been reached in tha t shor t time-frame,

e.g., PND 4. The lack o changes in in offsp ring on PNDs 14 and 21 no te on ly the

dams were continued on treatmen t were interpreted by the authors to indicate that

lac tational transfer did no t play a sign ificant role in maintaining thyroid homeostasis and

that changes seen at PND 4 were transient.

In a pubertal study, Zorrilla et al. 2009) treated groups o male Wistar rats 8 to

15 pe r group) with 0 3 30, 100 200, or 300 mg/kg /day triclosan in corn oil by oral

gavage from PND 23 to 53 31 treatmen ts). According to the authors, th e protoco l used

was that defined in the Endocrine Disruptor Screening Program E DSP) USEPA 2009)

to evalua te male pubertal effects from exposure to xenobiot ics. No visible signs o

‘ nly poster presented at the Society of Toxicology annual meeting in 2 9 was available for reviewNote that the Figure from which these data were obtained was not consistent with the text of the abstractof this poster that states that T4 levels were not affected in pups at any PND evaluated

4




Investigation of Potential Endocrine Activity of Triclosan September 4 9

tox icity were noted in any of the trea ted an imals over the course of treatment. No

treatment-related changes were seen in body weight gain g row th) or in term inal body

weigh ts at necropsy. Kidney and adrenal weigh ts were una ffected, while liver weights

were significantly increased in the 100, 200, and 300 mg/kg/day, a finding consistent

with all of the subchronic and chronic toxicity da ta for triclosan M olito r et al. 1992;

Eldr idge 1993; Auletta 1995; Molitor and Pershon 1993; Pershon and Molitor 1993; Yau

and Green 1986).

In the Zorrilla et al. 2 009) study, sign ificant dose-dependent decreases in the

mean serum total T concen trations were seen at 30, 100, 200, and 300 mg/kg/day with

no effect at mg/kg/day. Serum concentra tions of total T were reduc ed to

approximately 8 and 44 of control in the 30 and 100 mg/kg/day groups,respective ly, and to app rox imately 25 and 22 of control values in the 200 and 300

mg/kg /day groups, respectively. The lack of a clear monotonic dose response may be an

artifact of the protoco l. Two contro l groups were used and designa ted as Block andBlock 2 by the authors. Block contained the 0, 3, 30, and 300 n10 mg/kg /day dosegroups and Block 2 contained the 0 n1 5), 100 and 200 both n8 mg/kg/day dose

groups. The change in T leve ls between control groups was sign ificant ly different and,

the refore, the abso lu te change in T levels for treated groups was compared to the ir

respect ive controls. It is unc lea r from the Figures in the paper Fi gures 6A and 6B) tha t

the pe rcent of control fo r each dose group was de termined from the mean of both control

groups combined or to the mean of the respect ive control group to the tre ated groups.

Also in the Zorrilla et al. 2 009 ) study, no consistent , treatment-related changes in

total serum T concentrations were seen there was a significant inc rease in the

mg/kg/day group and a significant decrease at 200 mg/kg/day but T plasma

concentrations were not af fec ted in 30, 100, or 300 mg/kg/day dose groups). Also , no

tre atment-re lated changes in thyroid stimulating hormone TS H) were seen in any dose

group tested.

At the histo logical exam ination, no sign ificant changes were seen in the follicu lar

epi thelia l height in any dose group . Decreases in colloid area were seen in thyroid gland

sec tions but only in the 300 mg/kg/day dose group, which, according to Zorrilla et al.

2009), indicated a compensa tory re lease of active thyroid hormones from the collo id.




Investig ation Po ten tial Endoc rine Activ ity Triclosan Sep tember 4 9

Changes in thyroid colloid area in combination with an inc reased height of fo llicular cells

along with hype rtrophy and hyperplasia of the fol licu lar cells could indicate persistent

TSH stimulation Khan et al. 1999). However, no changes in fol licu lar heigh t or signs of

hypertrophy or hyperplasia were seen at any dose level. Further, leve ls were

decreased at doses of 30 mg/kg/day and higher but no changes in colloid morphology

were noted in rats receiving tric losan at doses less than 300 mg/kg/day. Also , consistent,

dose-related changes in levels o were not seen and TSH levels were not affected by

trea tment with tric losan. Morphological changes in co lloid area in the absence of

fol licu lar cell changes are of uncertain relevance.

2.1.2 Review of hum n studyIn contrast to the rat studies conducted by Crofton et al. 20 07), Paul et al. 2 009)

and o nill et al. 2009), triclosan, administered in too thpaste to healthy volunteers did

not result in an increase in thyroid hormone leve ls A llmyr et al. 2009). Twelve

volun teers 5 men and 7 women), who discont inued use of any triclosan contain ing

products two weeks prior to the initiation of the study , were instructed to brush tw ice

da ily using an over the counter too thpaste Colgate Total®) for 14 days. The

concentration of triclosan in the toothpaste was 0.3 w/w) and the amoun t applied was

standard ized 2 cm), which was estimated by the authors to resul t in a dose of swallowed

triclosan of 0.01 mg/kg/day. This dose was based on the estimates of intake of triclosan

from daily brushing B agley and Lin 2000). The subjec ts were ins truc ted to brush for

minutes per event and no toothpaste was to be swallow ed intentionally. The pro tocol was

designed to provide an estimate of the upper range of exposures to triclosan from the use

of toothpaste that could be expected in a normal human population .

In the llmyr et al. 2 009) study, plasma triclosan levels were measured before

the experiment and afte r 14 days of brushing with plasma concen trat ions of triclosan

both unconjugated and con jugated form s) ranging from 0.009 to 0.81 ng/g before

exposure to 26 to 296 ng/g after 14 days of brushing. Afte r exposure , the median

triclosan concentra tion in plasma was 54 ng/g, which was pproxim tely tim es higher

than the triclosan concentrations measured in serum samples in popula tions in Sweden

and Australia A llmyr et al. 2006. 2008). Based on this comparison , llmyr et al. 2009)

6




Invest igatio n of otential ndocrine Ac tivity of Triclosan Sep tember 4 9

concluded that the instruct ions for brush ing used in their study, e.g.. the duration and the

amount, overstated the amount of tric losan exposure from standard brushing procedures.

According to kine tic studies, approximately 70 of the tric losan in plasma is in

the form

of the sulphate or glucuronide conjugates (Sandbo rgh-England 2006), which are assumed

to be biologically inactive (A limyr et al. 2009).

After the exposure period, Ailmyr et al. (2009) measured plasma concentration s

of 43-hydroxycholestero1, free plasma concen tra tion of and TSH. No significant

changes in plasma concentrat ions of any of the thyro id hormones were seen compared to

baseline leve ls measured prior to ini tiation of treatment nor were any outside of normal

reference values. No sign ificant changes were seen in plasma concentra tions for 4f

hydroxycho les te ro l which is a cho lesterol metabolite and an endogenous marker for

CYP3A4 ac tiv ity K aneb rat t et al. 2008; Wide et a . 2008; Diczfalusy et a .2009). No

marked dev iat ion from the resul ts found for the entire group was noted in the individual

in this study with the highes t triclosan plasm a concentration of 296 ng/g, which is in the

range of plasma levels obtained after an oral dose of 4 mg triclosan (Sandborgh-Englund

2006).

2 3 Comparison among recent thyroid studies

Given that the above studies in rats were conducted by the same group, a number

of compa risons can be made that may be useful in assessing the relevance of these

findings to hum an health outcomes . In a comparison of the Crof ton et al. (2007) and Paul

et al. (2009 ) studies, both examined female Long Evans rats given triclosan at similar

concentrations but ages at first exposure were different (young weanl ing rats at 23 to 25

days of age v. sexually mature rats) and fo r diff erent dura tion s (4 days compared to 36

days). Despite the long er duration of dos ing and older females tested in the Paul et al.

(2009) study compared to tha t repo rted by Crofton et a .(2007), sign ificant decreases in

maternal levels were only seen in the 300 mg/kg /day dose group and were comparable

with a 34 v. 30 dec rease in the Crof ton et al. (2007) and Paul et al. (2009) studies,

respectively. These da ta wou ld ind icate that with an inc reased duration of treatment ,

levels were not further depressed. However, there was a significant difference in

response in the 100 mg/kg /day dose groups [28 v. 8 in the Crofton et al.(2007 ) and

7




nvestigation of Potential ndocrine Activity’ of Triclosan eptem er 24 2 9

Paul et al 2009) studies, respect ively] that may indicate tha t expo sure at a younge r age

would shift the dose- respon se curve with sign ific ant responses occur ring at lower doses;

however, no statistica l analyses were conducted to discern if the response in the 100

mg/kg /day dose group was sign ificant ly differen t from that in the 300 mg/kg/day dose

group in the Crofton et al 2007 ) study. The reduction in levels was comparable in

both studies for the 30 mg/kg/day group n on -significan t decreases of 7 v 8 ). These

differences may reflect na tural variabi lity due to the small numbers of animals tes ted [16

per group in the Croflon et al 2007) study; the number of an imals per dose group was

not given in the Paul et al 2009) poster] but does introduce uncertainty as to an estim ate

of a threshold for this effect.

When the resultsof

the Pau l et al 2009) and Crofton et al 2007) studies arecompared with those reported by orrill et al 2009), there are differenc es in responses.

In the Zorrilla et al 2009) study, significant changes in total serum concentrations

were seen in weanling male Wistar rats in the 30 100 200 and 300 mg kg day dose

group given triclosan for 31 days a pp rox imately 50 reduc tion in 30 and 100 mg/kg/day

groups and 80 reduct ion in the 200 and 300 mg/kg /day groups). In the Paul et al

2009) study a significan t decrease in total serum concen tration s was noted in adult

females only in the 300 mg/kg/day dose group a decrease of approximately 30 from

control va lues) given tri closan in the same manner for 36 days. The key differences in

the Zorrilla et al 2009) study and that conducted by Paul et al 2009) were the gende r

difference, the rat stra in tested , and the age at initia tion of dosing, which may not be a

de termining factor as noted in the compa rison between the Crofton et al 2007) and the

Paul et al 2009) stud ies men tioned in the preceding paragraph.

Differences in total serum concentra tions following triclosan tre atment were

also seen between the results of Crofton et al 2007) and Zorrilla et al 2009) studies in

which the animals were the same app rox imate age at the in itia tion of the study but the

du ration, gender, and species were different. The du ration of exposure, at least in

females, was not a factor, as noted above. Gender differences in rats are known to exist,

e.g., male rats may develop more thy roid tumors than female rats due to greater changes

in TSH or to complex interactions with tes tosterone USEPA 1998). However, TSH and

tes tosterone levels were una ffected at any dose tes ted in the Zorrilla et al 2009) study .




Investigation of Potential Endocrine Activity of Triclosan September 24 , 9

The levels of TSH are higher in male rats than in female rats, i.e., the demand on the

thyroid gland of the male rat is higher than in female rats, and chemicals that interfere

with thyroid hormone homeostasis would likely have more of an impact in male rats than

in females USEPA 1998 . Another factor is the difference in the rat strains tested;

differences in sensitivity have been noted for other endpoints, such as the production of

Leydig cell tumors in Fisher 344 rats or mammary carcinomas in Sprague Dawley rats

compared to other strains. However, all of these differences may also reflect inherent

variability in this assay due to the use of total bound and unbound concentration

rather than free as the metric.

The results in the rat studies can be compared to those reported in a study of

humanvolunteers. As noted above, serum concentrations of total

were reduced in

female rats at doses between 100 and 300 mg/kg/day Crofton et al. 2007 , while levels were significantly reduced in male rats at doses of 30 mg/kg/day and higher

Zorrilla et al. 2009 . No changes in serum concentrations were seen in volunteers in

the Ailmyr et al. 2009 study. There are a number of reasons for the lack of agreement

between the changes in 14 levels in the rat studies and the results reported by Alimyr et

al. 2009 . The most obvious reason is the difference in the dose of triclosan. In the ra t

studies, the dose at which a change in levels was significantly different from controls

ranged from 30 mg/kg/day to 300 mg/kg/day. The estimated dose to the subjects in the

Alimyr et al. 2009 study was estimated to be 0.01 mg/kg/day, a dose that could result

from typical use of triclosan containing toothpaste. Perhaps the more important reason,

as discussed in Section 2.3, is the differences in homeostatic controls in the ra t and

human. It is important to note that serum concentrations of the biologically active

thyroid hormone in both species, and TSH were not altered in either the Zonilla et al.

2009 study in ra ts or the Alimyr et al. 2009 study in human volunteers.

While it is impractical to establish a threshold in an experimental study, the data

in rats do demonstrate that there are administered doses of triclosan below which no

treatment-related changes in 14 levels were measured. Further, in the rat studies, total

bound and unbound 14 concentrations were measured, while only free 14 levels were

measured in humans. Use of free 14 as the relevant metric has been recommended by

DeVito et al. 1999 , who reported the summary of the working group on screening

9




Investigation of Potential Endocrine ctivityof Triclosan September 24, 2 9

methods for thyroid hormone disruption. Use of total 14 determinations was not

recommended by this working group as a screen for thyroid function but rather free 14

and TSR leve ls because changes in bound 14 could occur without changes of the

biologically available 14 levels DeVito et al. 1999 .

2.2 Comparisons to Clinical and Experimental Data

The safety of triclosan has been evaluated in studies with human volunteers

designed to assess tolerability and/or kinetics and has been extensively studied in

experimental animals primarily by the oral and dermal routes of exposure with acute,

subacute, subchronic, and chronic administration as reviewed by Rodricks et al. 2009 .

None of these studies was specifically designed to evaluate thyroid hormone levels or

thyroid function. Under the assumption that decreases in serum 14 concentrations could

lead to or be a marker for hypothyroidism as suggested by Crofton et al. 2007 and

Zorrilla et al. 2009 , a number of adverse human health outcomes could be hypothesized.

Thyroid hormones influence normal metabolic functions in most tissues in the body. The

consequences of decreased thyroid hormone levels can be manifested in a number of

ways, e.g., overt clinical signs, such as fatigue or subtle changes in biochemical

parameters, and the nature and extent of the effects could depend on the affected organ

and the timing or age at which hypothyroidism occurs. Therefore, clinical signs and

symptoms that could be the consequences of hypothyroidism were evaluated in these

clinical and experimental studies. The potential effects were grouped into four major

categories:

• Overt signs of toxicity and or changes in metabolic or biochemical parameters;

• Continued pressure on thyroid homeostasis resulting in the production of thyroidhyperplasia and neoplasia; and,

• Effects resulting from altered thyroid homeostasis during critical windows ofdevelopment:

o Neurobehavioral deficits resulting from in utero exposure, ando Alterations in reproductive competence resulting from exposure during

the sensitive developmental times to include neonatal and juvenile

periods.

10




Investigation of Potenti al ndoc rine c tiv ity of Triclos an September 4 9

2.2.1 Overt signs of toxicity and or changes in metabo lic or biochemicalparameters

A number of studies have been conducted to specifica lly evalua te hum an safety

and to lerability of triclosan in oral care produc ts DeSalva et al. 1989; Lucke r et al. 1990;

Safford 1991; Barnes 1991 a; Barnes 1991 b). eSalva and coworkers published a review

of triclosan safety that summarized the resul ts of cl inical stud ies of oral expo sure to

triclosan contain ing toothpas te or mouthwash products D eSalva et al. 1989). Although

individual studies were not identified , th is publica tion reported resul ts from 1,246

participants in clin ical studies in which toothpa ste or mouthwash with triclosan at

concentrat ions rang ing from 0.0 1 to 0.6 had been used for day to 12 weeks. Nodifference between contro l and expo sed popu lations with respect to hematology and

clinical chem istry findings were repor ted , including tests for live r and kidney func tion.Safford 1 991) evaluated hematolog ica l and clinical chemistry changes in 112

sub jects who brushed with toothpaste containing 0.2 triclosan for 65 weeks. Subjects

n=392 ) in the Barnes et al. 199la; 1991b) study brushed no rmally with 0.2 triclosan

containing toothpas te for 52 weeks fol lowed by a 13-week perio d without triclosan

containing too thpaste. In a study designed to eva luate the kinet ics of triclosan , 20

subjects were g iven increasing amounts of triclosan for 52 days; do ses increased from mg/day to 30 mg/day over a 2-week period fo r a tota l of 10 days, followed by 15 mg/day

for 30 days Lucke r et al. 1990). These studies evaluated changes in clin ica l biochemical

and hematolog ica l endpoints in humans and, in the case of Lucker et al. 1990) , urina ry

parameters. In all of these stud ies, no clinically re levant, sustained, trea tm en t rela ted

changes in hematological or clin ica llbiochemical parameters were observed in more than

2500 sub jects following the da ily use of toothpa ste or other oral products containing

0.01 to 0.6 triclosan for periods ranging from day up to 4 years.Similarly, in the numerou s expe rimental stud ies conducted there were no overt

clinical sign s of toxicity following acute, subchron ic or chronic administration of

triclosan at very high doses Rodricks et al. 2009). Further, there were no treatment-

re lated, con sistent effects on any hematological parameters or clin ica l chem istry non

liver) parameters, When present, alterations in enzymes associated with live r func tion

were found to inc lude decreases in cholesterol, consistent with the proposed mode of




Investigation Potential Endocrine Activity Triclosan September 24 2 9

action of tri los n as a PPARCL gonist in the liver, the main t rge t organ ident ified in all

of the numerous experimen t l studies Rodr icks et al. 2009 .

2.2.2 Production of thyroid hyperplusia and neoplasia

Chemica ls known to in terfere with t yroi hormone homeostasis in rats are

structurally diverse and interfere with thy roid hormone homeost sis by different

mechanisms USEPA 1998 . The general hypo thes is is that an alteration in the balance of

thyroid hormones. i.e ., decreased circulating leve ls due to either decreased production of

or increased metabolism and ex retio n of orT triggers, through feedback loops ,

the release of thyrotropin releasing hormone TRH from the hypothalamus, with the

resulting cascade lead ing to an increase in TSH release from the pitu itary. The sustainedstimulus by TSH on the thyrocy tes is to produce more thyroid ho rmones. The end result

is that in rodents, primarily rats, fol lowing the administration of the se chem ica ls, thyroid

hormone leve ls and can be decreased , resul ting in a com pensatory increase in

TSH in order to maintain and on en tr tion s at physiological levels, With

continued chemica l inte rfe rence, the elevated levels of TSH wou ld be sustained resul ting

in a persisten t signa l to the follicular ce ll to produce T thereby , promoting the stimulus

to hyperplasia and neoplasia in rats LJSEPA 1998 .

Free levels of or 14 are subject to rapid metabo lism and degradation . Unlike

humans , rats lack spec ific thy roid hormone binding globulin s and, therefore, lack the

reserve capacity to rele se thyroid bound horm ones in response to dec reases in free

serum thyroid hormone levels. The rat thyroid gland is at near m x imum stimulation in

order to m int in physiolog i l levels of thyroid ho rmone . The addition of a chemical

that r esults in decreased serum levels of and with a compensatory increase in TSH

would further stress a system that was near capacity . f the TSH stimulation was

sustained, hyperplasia and possibly prog ression to neopl si could resu lt in rats USEPA

1998 . This prog res sion by non-genotoxic modes of ac tion to thyroid tumors has not

been seen in humans USEPA 1998 .

In the rat study conducted by Zorrilla et al. 2009 no sustained treatment-related

altera tion s in or TSH levels were reported. Under the assumption that 31 days may

not have been su ffi ien t to see a sustained change that cou ld resul t in com pensatory




Investigation of Potential ndocrine Activity of Triclosan September 24, 2009

increases in TSH with resulting biological con sequences, other expe rim ental data for

triclosan were reviewed. Thyroid histop athology has been eva lua ted in several of the

subchronic and chron ic roden t toxicity studies. No adverse effects on the thyroid have

been no ted in any of these stud ies sugg est ing that the reduc tions of serum in the short-

term studies by Crof ton et al 2007) and Zorrilla et al 2009) would no t have lead to

altered thy roid histopa tho logy follow ing chronic exposure.

In those stud ies in which thyroid weights and/or histopa tho log ica l eva lua tion s

were conducted, there was no ev idence of an effect on the thyroid in subchronic studies

in mice at doses up to 900 mg/kg/day Trutter 1993), rats at doses up to 300 mg/kg/day

Goldsm ith and Craig 1983), dogs at doses up to 25 mg/kg/day L euschner et al 1970) or

Rhesus monkeys treated dermally with a 0.1 t riclosan solution daily D algard et al

1979; Parkes. 1979). No effects on thyroid weigh t or histopa thology were seen in

chronic/oncogenicity studies following administ ration of triclosan in the diet in mice at

doses up to 200 mg/kg/day Auletta 1995). rats at doses up to 15 mg kg day Y au and

Green 1986), hamsters at doses up to 250 mg/kg/day Chambers 1999) or baboons at

doses up to 300 mg/kg/day Drake 1975). Triclosan did not produce thyroid tum ors in

any species tes ted when administered at doses that were above those that were reported to

produce changes in leve ls in the short- term studies reported by Crof ton et al 2007),

Zorrilla et al 2009), and Paul et al 2009).

2.2 .3 Altered thyroid homeostasis during critical windows of developm en t:

2.2.3.1 Neurobehavioral deficits resulting from in utero exposure

It has been prop osed that hypothyroid ism in general, and hypothyroxinemia in

particular, in women during the first trimester of pregnancy could affect normal

developm ent of the feta l brain and lead to neurobehavioral deficits in child ren born to

these mothers M orrea le de Escoba r et al 2000 . Hypo thyroidism has been de fined by

Morreale de scob r et al. 2000 reviewed literature drawn primarily from studies in geogr phic l areaswith a high incidence of cretinism due to maternal iodine deficiency or that reported an association betweenwomen with maternal hypothyroidism defined as having TS H levels higher than the

percentile of the

normal range or hypothyroxinemi undefined as to extent and neuropsychologic l deficits in theiroffspring. In contrast, Soldin et al. 2003 found no evidence to suggest that in utero thyroid status asreflected by neonatal levels had an impact on the neurologic l disorders di gnosed in childhood. In theSoldin et al. 2003 study, to determine if there wa s an association between childhood neurobeh vior l




Investigation of Potential Endocrine Activity of Triclosan September 24 2 9

the American Thyroid Assoc iation as decreased free concentrat ions accompanied by

increased serum TSH concentration s suf ficient to disrupt thyroid hom eos tas is D eVito et

al. 1999 . Potentia l neurop sychological effects in offspring from maternal exposure to

chem ica ls may only occur at doses that produce sufficien t hypothyroid ism or

hypo thyroxinem ia in the mother, such that maternal transfer o to the fetus is

sufficien tly impaired D eVito et al. 1999). For example, decreases in levels 55 ) in

the early pos tna tal pe riod were not sufficient to alter synaptic transm ission in the den tate

gyrus of the hippocampus of rat pups G ilbert et al. 2002). Consequently , in the absence

of maternal hypothyroid ism or hypothyroxinemia, subsequen t neurobehav ioral defic its as

a result of impa ired thyroid funct ion are unlik ely to occur. According to DeVito et al.

1 999) change s in thyroid hormone levels and histopa tho log ica l changes would occur atlower doses than those requ ired to detect behavioral changes. The refore, changes in free

and TSH levels are recommended as a screen for neurobehav ioral potential rather than

developmental assays D eV ito et al. 1999).

When triclosan was administered to pregnan t females , decreases in levels were

only seen in the 300 mg triclosan kg day dose group and not in those females rece iving

30 or 100 mg kglday P aul et al. 2009). In this study , a transient decrease in levels

was seen only in pups born to dams in the 300 mg/kg/day dose group and on ly when

measured at PND 4 but not on PND 14 or 21 and no changes in maternal or fetal

levels were seen at materna l doses up to 100 mg/kg/day. Further, no al teration s in

or TSH levels were seen in hum an vo lunteers given triclosan c ontaining toothpaste that

resulted in doses relevan t to those tha t may result for the use of triclosan -con taining

products AIlmyr et al. 2009 . At doses of triclosan, such as those encountered as a result

of use of triclosan-containing consumer products, alterations in thyroid hormones are

unlikely to occur and con sequently, neu rob ehavioral effects are un likely to occur at doses

relevan t to potentia l human exposures.

deficits such as attention deficit hyperactivity disorder ADHD . learning disorders or autism that couldbe attributed to fetal thyroid hormone deficits neonatal levels were obtained from a neonatalhypothyroidism screening program All cases were diagnosed at medical school diagnostic clinics Nosignificant differences were found between neonatal levels of cases and controls for any neurobehavioralcondition

14




Investigation of Potential Endocrine Activity of Triclosan September 24 2 9

2.2.3.2 Alterations in reproduc tive competence resultingfrom exposure during thesensitive developmenta l times to include neonatal andjuvenile periods

Another potential concern is that hypothy roidi sm induced in the early neonatal

per iod can resu lt in effects on reproduc tive development and funct ion in male rats

manifested as alterat ions in sexual maturat ion, testicular and rep rod uct ive tract

developmen t and changes in gonadotrophin and steroid concen tra tions and by having an

impact on both Ser toli cells and Leydig cells Zorrilla et al 2009 . This top ic is

discussed further in Section 3

2.3 Relevance to Human Health Outcomes

The po tential for alte red levels seen in the rat stud ies to occur in humans and

have an adverse effect on health is dependent on the mode of actio n for these decreases in

levels and the relative suscept ibility to changes in homeostasis between species that

could result. There are a number of steps in the normal production of thyro id hormones

and the maintenance of thyroid homeo stasis that could be the targe ts of xenobiotics in

rats . Changes in ei ther the product ion of the metabolism o to or the binding

andlor the elim ina tion o from the circulat ion could impact homeo stasis and,

consequent ly , engage the normal physio log ical feedback loop to compensate for such

changes; the latter two gener al steps are media ted by liver enzymes.

In biochem ical and mechanistic studies, tric losan has been shown to induce

selected liver enzyme systems Mol itor et al 1992; Eldridge 1993; Molitor and Persohn

993; Persohn and Molitor 1993; Persohn 1994; Thomas 1994 . Triclosan produced an

increase in CYP AI or CYP A in mice Molitor et al 1992 and rats Molito r and

Persohn 1993 but not in hamsters Thomas 1994 .

The CYP A fam ily ofmonooxygenases catalyzes many reactions inc lud ing the

synthesis of cholesterol, steroids, and other lipids Sm ith et al 1998 . CYP3A mediates

the metabolic act ivatio n of uridine diphosphoglucu ronosy l transferase UDPGT Allmyr

et al 2009 . This could result in an increase in glucuronidation o after conjuga tion

with hepatic microsomal UDPGT, which could result in an increased clearance o in

‘The CYP3A family serves a common function across species to include metabolism of steroid and lipids.There are slight variations across species Carre t al. 2006 . The predominant forms are CYP3AI orCYP3A in rats; CYP3AI and CYP3A26 in dogs CYP3A6 in rabbits CYP3A64 in rhesus monkeys andCYP3A4 and CYP3A5 in humans Carret al. 2 6




Investigation of Potential Endocrine Activity of Triclosan September 24, 2 9

the b ile Saghir et al. 2008 , one potential way in which serum levels could be

reduced.

Triclosan’s effects on selected enzymes were assessed in rats Zorrilla et al. 2009;

Paul et al. 2008, 2009 . In rats in the Zorrilla et al. 2009 study, an increase in UDPGT

activity was seen in the highest dose group 300 mg/kg/day compared to controls.

Although the authors stated that the increase was no t statistically significant, UDPGT

levels were at least 2 to times that in the control group or the two lower dose groups

tested 3 and 30 mg/kg/day . Decreases in levels were noted at a dose of 30

mg/kg/day and higher; however, UDPGT activity was not elevated in the 30 mg/kg/day

group; the 100 or 200 mg/kg/day dose groups were not evaluated for UDPGT induction.

The authors noted that this type of inconsistent relationship between UDPGT inductionand levels has been reported previously by Craft et al. 2002 , de Sandro Ct al. 1992 ,

and Hood and Klaassen 2000 and was likely due to variability inherent in this assay.

Transcription of CYP is mediated by the human nuclear receptor hPXR

Ailmyr et al. 2009 and by PPARL agonists Klaunig et al. 2003; Molitor et al. 1992;

Molitor and Pershon 1993 . Triclosan was found to be a medium affinity ligand to hPXR

when tested in vitro in the human hepatoma cell line HuH7 transfected with human

PXR Jacobs et al. 2005 . However, no significant changes were seen in plasma

concentrations of 43-hydroxycholesterol, a cholesterol metabolite and an endogenous

marker for CYP3A4 activity in humans following use of triclosan containing toothpaste

compared to pre exposure levels Ailmyr et al. 2009 . The lack of an increase in a

defmitive marker of CYP3A4 activity in subjects in the Ailmyr et al. 2009 study would

indicate that at the triclosan exposures at environmentally relevant amounts, activation of

CYP3A4 and resulting biological consequences, such as the increased excretion of in

the bile is not occurring. The significance of these findings is relevant to the difference

in species response to triclosan. Induction of hepatic enzymes that may be involved in

thyroid hormone metabolism and catabolism may also be mediated by triclosan’s

demonstrated activity as a PPARa agonist, a mode of action that is not likely relevant to

humans Rodricks et at. 2009 .

Regardless of the specific mode of action, there is considerable evidence that

humans are more resistant to changes in circulating thyroid hormone levels than rats

16





because of the significant buffering capacity of thyroxine binding proteins TBG in

humans USEPA 1998 . There are also species differences in the plasma half-lives of

thyroid hormones. and are bound to plasma proteins, such as albumin, in most

mammalian species. Protein bound thyroid hormone is unavailable for metabolism and

serves as a buffer for changes in peripheral and levels. However, in addition to

albumin, some species, including humans and primates, have high affinity and binding globulins and a large percentage of and is bound to these proteins USEPA

1998 . In contrast, r ats and mice lack these binding globulins, and a smaller fraction of

and is bound to protein in rats and mice. Unbound or free and is available for

metabolism, resulting in faster hormone turnover in rodents than in humans, with plasma

half-lives of 12 to 24 hours and

to 9 days reported for

in rats and humans,respectively USEPA 1998 . Further, TSH levels are from 6 to 60 times higher in rats

than in humans USEPA 1998 . Therefore, due to the rapid hormonal turnover, the ra t

thyroid gland must work harder and is continuously stimulated in order to maintain and within physiological levels. Consequently, the rat thyroid gland would be more

susceptible to chemical perturbation of thyroid hormone homeostasis than the human

thyroid gland USEPA 1998 . A number of compounds have been shown to affect

thyroid hormone levels, including the PPARa-agonist, clofibrate, without an impact on

normal thyroid function in humans Visser et al. 1993 .

3.0 POTENTI L EFFE TS ON REPRODU TION AND DEVELOPMENT

3.1 Recent Studies in Mammalian Systems

Recent studies have been conducted to evaluate the potential for triclosan to

impact the reproductive developmental system. These have included studies conducted

following in vivo Kumar et al. 2009; Zorrilla et al. 2009 or in vitro Kumar et a l. 2008;

James et al. 2009; Gee et al. 2008; Chen et al. 2007; Ahn et al. 2008 administration of

triclosan.

As noted above for the changes in serum thyroid hormone concentrations,

changes in levels of circulating hormones associated with male or female reproductive

systems in rats serve as a screen for the potential of a xenobiotic to have an adverse

impact on reproductive developmental competency in humans. Such changes in these

17




Investigation of Potential ndocrine Activity of Triclosan September 24, 2009

screening assays should be viewed in parallel with in vivo tests for

reproductive/developmental effects, when conducted as they have for triclosan, to

determine if changes in hormone levels measured in these screening studies were

sufficient to affect function.

3.1.1 Review studies conducted in vivo

Two studies have been conducted in the same strain of male rats (Wistar) in

which many of the same parameters were evaluated with conflicting results (Kumar et al.

2009; Zorrilla et a . 2009). These studies are discussed in this section.

Kumar et al. (2009) investigated potential antiandrogenic effects in male Wistar

rats administered triclosan in phosphate buffered saline via gavage once a day for 60 daysat concentrations of 0, 5, 10, or 20 mg/kg/day (n=8 pe r group). Treatment was initiated

when the animals were 10 weeks old, with s crifice occurring 24 hours following the

final treatment. As described below a number of endpoints were evaluated; however, for

the majority of these analyses only the control and high dose group were tested and for a

number of those, testes from the control or the high dose group were homogenized and

the pooled samples tested.

Kumar et al. (2009) reported a significant decrease in the weight of testes and all

sex accessory tissues evaluated following administration of 10 or 20 mg/kg/day bu t not in

the 5 mg/kg/day, compared to controls. The authors reported significant decreases in the

enzymatic activity of both 3 j3-hydroxysteroid dehydrogenase (3 13-HSD) and I 7j3-

hydroxysteroid dehydrogenase 7f3-HSD) in the two highest dose groups with decreases

of 27 and 39 in 313-HSD in the 10 and 20 mg/kg/day dose groups, respectively, and

decreases of 31 and 46 in 7J3-HSD, respectively. Significant decreases in the

following endpoints were reported in the 20 mg/kg/day dose group (the only treatment

group evaluated): serum levels of leutinizing hormone (LH) (38.5 ), follicle stimulating

hormone (FSH) (17 ), pregnenolone (31 ), testosterone (41 ), and cholesterol (35 ).

Decreases were also seen in: daily sperm production (DSP) (34 ); mRNA for all genes

The methods section reports that rats were s crificed by cervical dislocation under ether anesthesia.However, the hormone serum concentrations were said to be determined from blood collected fromdecapitated animals by cardiac puncture. Typically truck blood is collected from decapitated animals andcardiac puncture if performed on animals euthanized

18




Investigation of Potential Endocrine Activity of Triclosan eptember 24, 9

evaluated P45Oscc. 313-HSD, l7f3-HSD. and P45 c androgen receptor AR and

steroidogenic acute regulatory StAR ; levels of protein for AR and S tAR protein all of

which were from pooled samples .

Histopathology examinations were conducted for the control and 20 mg/kg/day

dose group in the Kumar et al. 2009 study. Reduced sperm density was noted in the

lumina of the epididymal tubule as well as malformations of the vas deferens lumen

showed presence of sterocilia detached from the epithelium and presence of eosinophillic

bodies and degenerated and empty folliculli in prostate tissues. No noticeable

histopathological changes were reported in the seminal vesicles of the 20 mg/kg/day

treated rats.

Several questions arose in the review of the Kumaret

al. 2009 study

that,when

considered with all of the other experimental evidence, cast considerable uncertainty as to

the interpretation of these results. The authors noted that the reason for reporting only 20

mg/kg/day group for many parameters was because of similar responses in 10 mg kg

group; however, as noted above, the method section clearly stated that for all but two of

the analyses only the high dose group was evaluated. The methods section describes five

different analyses in which the testes were either fixed in paraffin sections analysis of

StAR protein or were homogenized and pooled niRNA to evaluate gene expression

using RT-PCR, levels of AR and StAR protein using Western blot analysis . It is no t

entirely clear from the protocol description if each whole testes was homogenized with a

sample drawn from each for an n=8, as is may be the case for the analysis of DSP, or as is

more likely the case for other assays, the testes of all animals were homogenized fo r an

ntl and then replicates of that pooled sample were evaluated. In the latter case n1 with

or 4 replicates , the statistical methods applied, i.e., Anova and student’s t-test, may be

inappropriate because what is being assessed is the variability in the test method not the

variability among test subjects.

Another concern is because of the various techniques required for these analyses,

i.e., each homogenization procedure was different requiring different reagents and

protocols, obviously with two testes per animal, two different procedures could have been

performed. However, with five separate analyses, as noted above, it is questionable that

19




Investigation of Potential ndocrine Activity riclosan September 4 2009

one testes from each of the eight males in the control or high dose groups could have

been eva lua ted in the same assay. Consequently , considerable uncerta inty remains.

Another question deals with pre-and pos t-treatmen t body weights in these

an imals. The final body weights of the male Wistar rats in the contro l and treated groups

appeared to be much lower than the average body weight of this stra in of rat. The

ave rage body weight of a male istar rat at 10 weeks of age is approximately 373 grams

Derelanko and Hol linger 2002 , which is grea ter than the ave rage body weight of the

con trol animals 168 ± 6.3 g repor ted by Kumar et al. 2009 . At 18 to 19 weeks of age,

male Wistar rats should weigh approximately 500 grams Derelanko and Hollinge r

2002 ; however, the body weigh ts of the control an imals in the Kum ar et al. 2009 study

averaged app rox imately 187 grams. While it is unclear how these differences may haveimpacted the resu lts, it does raise questions regarding the age of the animals tested and

the methods and results prov ided by Kumar et al. 2009 .

Very different resu lts were repor ted by Zorrilla et al. 2009 who investigated

many of th e same endpo ints. Zorrilla et al. 2009 conduc ted a study in male Wistar rats

to determine the potential effects of triclosan on pubertal development and on indices of

reproduc tiv e competence. Animals were administered triclosan da ily via oral gavage in

corn oil at doses of 0 3 30, or 300 mg kg day Block n1 or 0 100 or 200

mg/kg/day Block 2 n=1 5 for contro ls and n’ for treated groups Dosing began on

PND 23 and continued to PND 53, when necrops ies were performed.

Triclosan did not affect normal growth; body weights were approximately 300 g

at necropsy. Administration of triclosan did not affect the age of preputial separation at

any dose tested. Nor did triclosan af fec t the growth of reproduc tive tract organs; weights

of the testes, ven tral prostate, seminal vesicle, epididymides. and levator ani

bulbocavernosus muscle in the treated groups were com parab le to controls.

No consisten t, dose-related change in testosterone was measured. While a

signi ficant decrease in serum testosterone concen tra tion was observed in the 200

mg/kg/day group compared to the combined con trols, testosterone levels were not

decreased in the 300 mg/kg/day group. Nor were there sign ificant dose-related decreases

6When the values in the control groups for an endpoint under consideration were no t statistically

significantly different the controls were combined and results in the all treated groups were compared tothe mean of the control values

20




Investigation of otential ndocrine Activity of Triclosan September 24 , 2009

in serum androstenedione a testosterone precursor, or in serum or pituitary LH or

prolactin PRL) in treated groups compared to controls, indicating that the hormonal

triggers to the production of testosterone by Leydig cells were not affected.

Histological examination did not reveal any significant treatment-related lesions

or alteration in either the tes tes or the epididymides. The authors reported that a few

animals in the high dose group had multinucleated giant cells within the seminiferous

tubule epithelium; however, these changes were characterized as minimal and did not

correlate with testosterone levels or testes weight in the individual animals.

Both Kumar et a . 2009) and Zorrilla et al. 2009) evaluated changes in LH and

testosterone concentrations both indicators of a potential impact on steroidogenesis.

Kumar et al. 2009) reported significant decreases in LH, testosterone and testes weightfollowing administration of 10 and 20 mg/kg; however, these results are in contrast to

those reported by Zorrilla et al. 2009). No significant changes in testes weight or LH

serum or pituitary were reported by Zorrilla et al. 2009) at any dose tested up to 300

mg/kg/day). While a significant decrease in serum testosterone concentrations was

observed by Zorrilla et al. 2009) in the 200 mg/kg/day dose group, no significant change

was observed in the high dose group 300 mg/kg/day) and the changes observed in all

dose-groups were not dose-related. In addition, Zorrilla et al. 2009) did no t see any

histopathological changes in the reproductive tissues examined. More importantly,

Zorrilla et al. 2009) reported no change in the age of onset of preputial separation, an

indicator of the onset of puberty, which would have been expected if the changes in

serum hormones, i.e., testosterone were impacted.

In comparison, the range of doses administered was different, with a much

broader range of doses administered by Zorrilla et al. 2009) 3 to 300 mg/kg/day)

compared to Kumar et al. 2009) 5 to 20 mg/kg/day). The l ife stage or window of

exposure was also different. Zorrilla et al. 2009) administered triclosan to weanling rats

during the period of pubertal development PND 23 to 53), while Kumar et al. 2009)

administered triclosan to adult rats starting at 10 weeks of age. The duration of exposure

was different with a 31-day exposure in the Zorrilla et al. 2009) study and a 60-day

exposure in the Kumar et al. 2009) study. However, these differences are unlikely to

explain the considerable differences in response between the two studies.

21




Investig ation of Po tential ndocr ine tivity of Triclosan ep tember 24 2 9

The comparison of these resu lts calls into question tho se results repo rted by

Kumar et al. 2009 , especially since the an imals in the Zorril la et al. 2009 study were

administered triclosan during the per iod of development of sexua l maturity, typically

considered a sensitive window of exposure. The increased dura tion of exposure would

not be expected to result in such a difference in the dose respon se in these studies .

Because of the rapid metabolism and elimination of tric losan with lit tle potent ial for

bioaccumulation , the longer du rat ion of exposure should not have been a factor , as noted

above for evaluation of thyroid hormone levels see Crofton et al. 2007 and Paul et al.

2009

Kum ar et al. 2009 concluded that the effects observed in their study indicated

that tr iclosan may pose a hazard to human health by acting through var ious mechan ism sthat result in endocrine disrupt ion. As this relates to the reproductiv e/deve lopmental

system, no evidence of an impact on this organ system in animals has been demons tra ted

after chronic exposure in rats Yau and Green 1986 , in a two genera tion reproductive

study in rats M orse th 1988 , or in developmental studies in rats Schroeder and Daly

992a; Denning 1992 or in mice Christ ian and Hoberman 1992 , hamsters Piekacz

1978 or rabbits Schroeder and Daly 1992b .

3.1.2 Review of in vitro studies

In vit ro s tudies have been conduc ted with tric losan in pr imary Leydig cells

Kumar et al. 2008 , in cultu red sheep placental tissues James et al. 2009 , and

immortalized non target cells Chen et al. 2007 or cancer cell lines Ahn et al. 2009; Gee

et al. 2008 . In vitro tests are useful for screening purposes and for form ing hypotheses

on the mode s of action of a chemical that can then be tes ted in more robust in vivo

systems. Interpretation of these resu lts and extrapolation of these results to be predictive

of in vivo responses should be done with care and without extend ing this interpretation

beyond what the da ta are capab le of providing. In particular, the resul ts in immortalized

cancer cell lines are of uncertain re levance because of the potentially altered genomic

responsiveness to xenobiotics Gentry et al. 2009 . These in vitr o data are presented for

completeness but mus t be considered in concert with the sho rt- term in vivo studies

assessing the same endpoints, as discussed in Section 3 1 1 and , more importantly , the

22




Investigation of Potential Endocrine Activity of Triclosan September 24, 2009

results of long-term experimental studies, as discussed in Section 3.2. Further, only those

aspects of studies that evaluated the potential of triclosan to affect endocrine but no t non-

endocrine signaling systems are discussed in this section.

3.1.2.1 Primary Cell Lines

Kumar et al. 2008) conducted an in vitro study in Leydig cel ls from male Wistar

rats in order to assess potential antiandrogenic effects of triclosan. The isolated cells

were incubated with 0. 0.001, 0.01, 0.1, 1, or 10 tM triclosan for 2 hours in the presence

or absence of LH 100 ng ml). Changes in testosterone biosynthesis, cell viability,

adenylyl cyclase activity, cAMP production, P 5 c 3f3-HSD, 1713-HSD and StAR gene

expression, and activity of three enzymes P45Oc 3-HSD and 17f3-HSD) involved intestosterone biosynthesis were measured.

No significant reduction in cell viability, cellular proliferation an indicator of

cytotoxicity) or alterations in cellular morphology were noted at any concentration tested

other than the highest tested 10 tiM). A concentration-related significant decrease in

LH-induced production of testosterone by Leydig cells was measured at concentrations

tested from 0.01 to 10 jiM. In the absence of LH, no impact on testosterone by triclosan

administration jiM), compared to untreated controls, was reported. The authors also

reported a significant decreased expression and activity ofP 5 i

3f3-HSD, 17J3-HSD

and expression of StAR protein at concentrations of 0.01 jiM and greater, in the presence

of LH stimulation. These data suggested a decreased delivery of intermediates in the

biosynthesis of testosterone contributing to the decrease in testosterone synthesis noted.

In a separate experiment, cells from both triclosan-treated and vehicle controls

were treated with forskolin an adenylyl cyclase activator) or SQ22536 an adenylyl

cyclase inhibitor , a lone or in combination with triclosan 0.00 to 10 jiM) for 2 hours.

Kumar et al. 2008) reported that triclosan produced a dose-dependent decrease in the

activity of adenylyl cyclase and cAMP at concentrations from 0.01 jiM to jiM no

further decrease in activity was noted at the highest triclosan concentration).

Administration of forskolin increased testosterone production, which was returned to

control values LH-treated only) with jiM of triclosan; however, triclosan did not further

decrease testosterone levels produced by SQ22536. The authors suggested that the mode

23





130 days of gestation 3 sheep , with term being approximately 147 days. Placental

tissue was homogenized and cytosolic sulfonation and microsomal glucuronidation

fractions isolated and incubated with triclosan at final concentrations of 0.1 to 10 tM for

approximately 15 minutes. Triclosan was a substrate for sulfonation with an apparent Km

of 1.14 and apparent Vmax of 160 pmol/minlmg protein. There was no detectable

glucuronidation in sheep placental microsomes.

Sheep placental sulfotransferases formed only estradiol-3-sulfate and no t 7J3estradiol-sulfate suggesting that the sheep placenta contained SUET I a homolog of

human SULT1EI. Addition of triclosan at concentrations ranging from 0 to 2.5 nM

altered the kinetic constants for estrogen sulfotransferase resulting in a dose-dependent

increase in apparent Km for estradiol 1 nM and a decrease in the apparent Vm.Triclosan also inhibited the sulfonation of estrone 2 nM in a dose dependent manner.

Inhibition was considered to be primarily competitive, with triclosan competing and

blocking the binding of estradiol and estrone to the active binding site on the enzyme.

The authors suggested that changes in extent of sulfonation of estrogen or estrone,

which are the predominant forms delivered to the fetus where the active steroid is

released, could result in an impact on successfully maintaining pregnancy. This outcome

is highly unlikely at environmentally relevant exposure to triclosan for a number of

reasons: 1 the role of placental estrogen sulfotransferases in the maintenance of

pregnancy in humans is not known with certainty James et al. 2009 ; 2 competitive

inhibition does no t mean a total cessation of the production of estrogen or estrone sulfate

conjugates and it is not known what level of inhibition would be outside of the

homeostatic range or the ability to deliver “enough” of the conjugate to maintain a

normal pregnancy, if such were necessary; 3 this in vitro analysis tested a specific

estrogen to triclosan ratio and it is unclear what that ratio would be with normal

physiologically levels of estrogen compared to concentrations of triclosan found in

human populations the higher the estrogen to triclosan ratio, the less the expected

competitive inhibition ; 4 at least in the humans, redundant sulfotransferases are

operative, e.g., SULT1A1 and SULT2A1 Stanley et al. 2001 ; and 5 because

detoxification by glucuronidation and sulfonation, primarily in the liver, a re the major

metabolic pathways in all species, in particular in the human liver Wang et al. 2004 , the

25




Investigation of Potential Endocrine ctivity of Triclosan September 24, 2009

systemic bioavailability of free triclosan to interfere with placental estrogen metabolism

at environmentally relevant triclosan exposures such that an impact on the fetus is

unlikely. Most importantly, if triclosan were actively interfering with estrogen transport

into the fetus, then administration of triclosan in vivo should result in an impact on

successful pregnancies; however, as discussed in the following section. there is no

evidence of an impact on reproductive performance or in the ability of rats to carry

fetuses to term Morseth 1988). While the observation that triclosan can be a competitive

substrate for sulfotransferase enzymes is not surprising given that sulfate and glucuronide

conjugates are the major detoxifying metabolites in all species Wang et al. 2004), a

significant impact on reproductive/developmental function at triclosan concentrations

detected in human populations is unlikely.

3.1.2.3 Immortalized or ancer Cell Lines

Chen et al. 2007) have developed a protocol that is designed to maximize any

potential effect of an antagonist, even if in an in vivo system the compound would be a

weak antagonist at the anticipated concentrations. In this in vitro study with human

embryonic kidney HEK) 293 cells, a stably transfected cell line that lacks critical steroid

metabolizing enzymes, the androgenic/antiandrogenic activity of triclosan was evaluated

in a cell-based human AR mediated bioassay. The cells were treated with or 10 .tM of

triclosan alone, testosterone alone 0.125 nM), or a combination of testosterone and

triclosan at these designated concentrations.

No cytotoxicity was reported when triclosan was tested alone at 10 iM or in

combination with 0.125 nM testosterone. No statistically significant differences in cell

proliferation were noted. Triclosan at a concentration of 10 jiM inhibited transcriptional

activity of testosterone by more than 92 , and by 38.8 at a concentration of 1.0 pM .

However, details of this assay were no t provided. Androgenic activity was not exhibited

at concentrations up to 10 pM in the absence of testosterone.

A number of aspects of the Chen et al. 2007) study limit it’s usefulness to be

predictive of triclosan effects in humans at environmentally relevant exposures . The cells

employed in this bioassay lacked critical steroid metabolizing enzymes and were

transfected with PCDNA6 hAR and an MMTV Luc.neo plasmid containing a luciferase

26




Investigation of Potential ndocrine ctivity of Triclosan eptem er 24 2 9

repor ting gene. The resulting cells are reported to be highly respons ive to endogenous

steroids and synthetic compounds. The authors stated tha t the concentra tion of

testosterone used in the assay was re lat ively low 0.125 nM) compared to the levels of

testos terone that the authors reported as mean circulating concen tration in humans 3.5 to

35 nM) C hen et al 2007). The authors noted that because only app rox imately 2 to 3

of circulating testosterone is free and cons ide red bioactive this low testos terone

concen tration was assum ed to be biolog ica lly relevan t; however, in the whole animal free

and bound testosterone are in dynamic equilibrium such that depletion of free

testos terone can be compensated by the release of bound testos terone to maintain

homeostasis. The authors noted that at this testosterone concentration, it is likely that

an tagonist ac tivity would be enhanced, which is an attribute useful for screen ing assaysbut of unce rtain quantitativ e re levance to normal in vivo behavior. The autho rs stated that

it was clear that the relative binding efficiency of triclosan for ARs w s orders of

magnitude be low that of the natural ligand tes tosterone). The refore, the respon ses of

this iso lated cell system are likely to be different than tha t of cells in normal

physiolog ica l situation.

In a study by Ahn et al 2008), an in vitro assay was conducted with recombinan t

cells that contained stably transfec ted estrogen E R) receptors to eva lua te the potential

biolog ical act ivity of tr iclosan. In the cell-based ER-med iated assay conduc ted in

recombinan t human ovarian cancer cells con tain ing a stab ly integrated R a responsive

firefly luc iferase reporter plasm id) , cells were incubated with one of three concen trat ions

of triclosan 10 nM, or 100 .tM) with or withou t I tiM estradiol for 24 hours. Triclosan

exhibited a concen tratio n dependent decrease of E2-dependen t repo rter gene exp ression

in the presence of estrad iol , with 50 inhibition observed at a concentra tion of lu

20 at 10 nM and 80 at 100 .iM). When tested alone , triclosan exhibited no

estrogenic ac tiv ity at any concen tration in the absence of estrad iol.

Gee et al 2008) conducted an in vitro assay with MCF7 McGrath human breas t

cancer cells to eva luate the ability of triclosan to bind to estrogen E Ra, ERJ3 receptors

or in the androgen responsive LTR-CAT reporter gene in Si mouse mammary tum or

cells in competitiv e binding assays . Assays were conducted to evaluate the ability of

triclosan to bind to estrogen- respons ive or androgen- respons ive reporte r genes and for

27




Investigatio n of Po tential ndocrine Activ ity of Triclosan eptember 24 2 9

triclosan’ s ability to increase the growth of estrogen- or androgen-responsive

immortalized breast cance r cells . In these assays, either estrad iol or testosterone was

added to the cells and the amount of triclosan, expressed as molar equ ivalents that could

effectively compe te for the specific bind ing site s was assessed . Triclosan was added to

these cultured cells at levels tha t exceeded endogenous es trad iol or testosterone levels, on

a molar basis, ranging up to 10,000 .000 times the concentration of the normal substrate.

Triclosan was able to inhibit estradiol binding to the ER in the cytosol ofMCF7

cells. MCF7 cells were incubated with 0.4 nM estradiol and the exten t of inhibition of

binding was determined using increasing concen trations of triclosan. Binding of estradiol

to the ER receptor could be inhibited by triclosan; however, triclosan concentrations

requ ired to do so were expressed as a molar excess of 100,000- to 10,000,000- fold the

concentration of triclosan to estrad iol that resu lted in an approximate 25 and 72

inhibition, respectively. When eva lua ting recombinant human ERa and ERI3, rather than

cytosolic ER, 63 inhib itio n was achieved at 100,000 molar excess of triclosan.

Comparing the ab ility of triclosan to inhibit recombinant ERa and ERf3, at 10,000 -fo ld

and 20,000-fold molar excesses, greater inhibi tion was observed with ERa 22 and 34 ,

respectively) than ER3 0 and 12 , respec tively) . riclosan was also able to inhibit the

stimulation of the es trogen-responsive ERE-CAT repor ter gene in these cel ls , but only at

100,000 fold molar excess concentrat ions of tri closan , compared to estradiol.

Separate assays to eva luate triclosan’ s ability to bind to the AR receptor and

regulate androgen responsive repo rter genes were conducted with recombinan t lig and

binding protein of rat AR and stably tran sfected androgen sens itive reporter genes L TR

CAT) in Si 15 +A mouse mammary tumor cells , respe ctiv ely G ee et al. 2008). In the

competitive binding assay with the recombinan t rat AR protein , triclosan inh ibi ted

testosterone bind ing by an average of 49 at 1000-fold molar excess and 77 a t 10,000-

fold molar excess. In mouse mammary tumor cells , triclosan was able to inhibit the

induction of the androgen responsive LTR-CAT repor ter gene, but only at concentrations

that were 100-fold molar excess of testos terone.

While these in vitro studies suggest potent ial activity of triclosan at certain

receptor sites, it must be considered that the cells used in some of these experim en ts were

conducted in inunortalized or cancer cell lines that are no t expected to respond in a

28




Investigation of Potential ndocrine tivity of Triclosan eptember 24 2 9

simila r manner to no rmal or prim ary cells entry et al 2009 . In add ition, cell lines,

such as those derived from the kidney, are not the target organ of concern and may no t

behave in a manner similar to cells from the appropr iate organ . The concent rations of

triclosan and endogenous steroids used in some of these assays i.e., Chen et al 2007

were selec ted by the au thors to achieve a maximum response and are not similar to those

expected in v vo Therefore, these in vifro results must be interpreted with caut ion when

extending them to the whole animal. It should be noted tha t no effects on reproduc tion or

deve lopment were repor ted in the battery of tests for triclosan conducted in vivo in rats at

doses of triclosan ranging from 15 to 150 mg/kg in a two genera tion reproductive study

Morseth 1988 or at doses of 15 to 300 mg/kg /day in one gene rat ion developmental

stud ies Schroeder and Daly 1992a, Denning 1992 . In addition, no developmentaleffects have been no ted in mice administered doses up to 25 mg/kg/day Christian and

Hoberman 1992 , or in rabbi ts administered doses up to 50 mg/kg/day Schroeder and

Daly 992b or in hamsters administered doses up to 80 mg/kg/day P iekacz 1978

3.2 omparison of New Studies to Other Experimental ata

Recent in vivo studies Kumar et al 2009; Zorrilla et al 2009 conduc ted in male

Wistar rats at different life stages adul t versus prepube rtal focused on the po tential for

triclosan to impact endocrine funct ion related to reproduc tive competence. The results of

these studies differed in their conclus ions. Fur ther, a comparison of these results with the

results from a battery of studies conducted in experimental an imals to determine the

po ten tial safety of triclosan is provided to put these recent studies in perspective.

Kumar et al 2009 suggested that the combined resu lts from their studies in

Wistar rats indicated that exposure to triclosan cou ld result in an impact on several key

steps involved in the biosyn the sis of testosterone , which in turn could have an impact on

reproductiv e func tion or deve lopment of offspring . However, the results repo rted by

Kumar et al 2009 were not dupl icated in developing male rats of the same strain, as

reported by Zorrilla et al 2009 . Even if the adult Wistar ra t were more sensitive to the

changes reported by Kumar et al 2009 , Zorrilla et al 2009 administered doses

app rox imately 15-fold greater 300 mg/kg/day than those administered by Kumar et al

29




nvestigation of Potential Endoc rine Activ ity of Triclosan eptember 4 9

2009) and still reported no significant change in critica l parameters, including

testos terone and LH concentrations , fol lowing triclosan adm inistration.

According to Kumar et al. 2009 ), signs of potential an tiandrogenic effects, e.g.,

dec reases in testosterone , in male rats occurred at low doses; however, the overall

toxicity database for tric losan compr ised of rep roduct ive/developmental studies and

chronic studies do not support effects on rep rod uct ion or deve lopment in this low-dose

region. In a two-genera tion reproductive study conduc ted by Morseth 1988 ) in

CRL:CD SD Br rats, no significant treatm ent -related effec ts were observed in an imals

administered triclosan in the diet at doses rang ing from 15 to 150 mg/kg /day from 10

weeks of age prior to mating with exposure cont inued throughout mating and then

ge station and lactation for females. No impact on reproduc tive pe rformance in eith ermales or females was repor ted nor was there any ev idence of toxicity during the growth

phase of ei the r generation.

The Kumar et al. 2009) resu lts are inconsisten t with Zorrilla et al. 2009), as

well as the results from a classic two-genera tion rep roduct ive study, bo th of which

administered much higher doses to the animals than those administered by Kumar et al.

2009). It is important to note tha t the majority of the parameters evaluated by Kumar et

al. 2009) are expected to have some biological variabili ty because of normal

compensatory mechanisms that maintain many phys io logical and biochemical param eters

within normal ranges and without impairm en t of function. Compar ison of the Kumar et

al. 2009) results with tho se from the reproductive, deve lopm en tal , subchronic and

chronic triclosan studies conducted in rats, as well as other species, suggests that if

triclosan was hav ing an impact on the pa rameters measured by Kumar et al. 2009) in this

low dose region, these changes were not ex tensive enough to resu lt in any impairment in

reproductive func tion or deve lopm en t following chronic exposure or exposure during a

critical windows of exposure.

3.3 elevance to uman ealth

Recent in vivo Kumar et al. 2009; Zorril la et a . 2009) and in vitro James et al.

2009; Chen et al. 2007; Gee et al. 2008; Ahn et al. 2008) studies have been conducted to

screen for the po ten tial for triclosan to have an impact the endocrine system. As noted,

30





the Kumar et al 2009 study the authors suggested that triclosan could be an endocrine

disruptor and affect reproductive systems; however, these results were not confirmed in

the study conducted by Zorrilla et al. 2009 .

While in vitro studies have been conducted to evaluate the potential for triclosan

to inhibit hormones, enzymes or receptors critical to steroidogenesis or reproduction

Kumar et al. 2008; Chen et al. 2007; Ahn et al. 2008; Gee et al. 2008 , these results must

be interpreted with caution. In many cases, concentrations of endogenous compounds in

the in vifro system were no t comparable to physiological conditions. In addition, some of

the studies were conducted in immortalized or cancer cells, which do not always have the

same active genes or react in a similar manner to normal cells Gentry et al. 2009 . For

many of these assays, the results were meant to be used as a screen, requiring additional

testing in the whole animal to confirm results, if such were not already conducted unlike

triclosan.

As noted above, the lack of reproductive developmental effects reported in

several triclosan studies in experimental animals do not support the potential for triclosan

to result in adverse reproductive effects in rats or humans. More importantly, humans

have high levels of naturally occurring estrogen and testosterone. As with thyroid

hormones, humans have a critical buffering capacity to bind these hormones and act as a

reserve to release free biologically active estrogen or testosterone, as well as critical

feedback loops involving the pituitary-hypothalamic axis to respond to decreased or

increased levels of estrogen or testosterone. Both the capacity in humans to bind and

release these hormones, with a much higher capacity in humans than in rodents, and the

feedback loops act to maintain homeostasis As with the thyroid, changes in hormone

status that could occur with exposure to a xenobiotic can be compensated for in humans

by these active systems that maintain homeostasis, especially at environmentally relevant

exposures to triclosan.

4.0 SUMMARY AND CONCLUSIONS

Crofton Ct al. 2007 and Zorrilla et al. 2009 reported dose-dependent decreases

in serum levels in rats administered triclosan in the diet for 4 days at doses of 100,

300. or 1000 mg/kg/day Crofton et al. 2007 or after 31 days of treatment at doses of 30

31




Investigation of Potential ndocrine ctivity of Triclosan September 24, 2 9

to 300 mg/kg/day Zorrilla et al. 2009 when administered to young weanling rats. No

consistent changes in levels were seen and TSH levels were unchanged after 31 days

of treatment Zorrilla et al. 2009 . Zorrilla et al. 2009 reported that there were no

treatment related changes in the follicular epithelial height in any dose group following

exposure to triclosan but decreases in colloid area colloid depletion were seen in thyroid

gland sections. but only in the high dose group 300 mg/kg/day . DeVito et al. 1999

stated that changes in histology, in particular changes in follicular structures were less

impacted by confounding than serum thyroid hormone levels and perhaps a better

indicator of alterations in potential thyroid function. Morphological changes in colloid

area in the absence of follicular cell changes are of uncertain relevance.

In contrast to the animal studies, Ailmyr et al. 2009 did no t find significantchanges in thyroid hormones in the plasma of volunteers after using triclosan containing

toothpaste. As noted above, no clinical signs of toxicity or changes in various clinical or

biochemical parameters were seen in human volunteers following the use of triclosan

containing products.

The weight-of-the evidence of all of the triclosan data is that in the rat model,

triclosan can affect circulating levels of total at high doses. While changes have

been seen in rats, signs of either hypothyroidism or other effects on the thyroid, e.g.,

changes in thyroid weight, or signs of hypertrophy or hyperplasia, have no t been seen in

several species administered triclosan for subchronic and chronic durations at doses

comparable to those administered in the short-term studies reported by Crofton et al.

2007 and Zorrilla et al. 2009 . A number of compounds including the PPARct-agonist,

clofibrate, a cholesterol-lowering drug, affect thyroid function in rodents without a

similar impact in humans. Moreover, humans have both sensitive feedback mechanisms

and extensive binding capacity, both of which maintain thyroid hormone levels with a

homeostatic range. Because of the robust nature of the human thyroid hormone system,

that is, resistance to changes in thyroid hormone levels, humans are expected to be less

sensitive to these changes than rodents. This conclusion is reinforced by the results of

Alimyr et al. 2009 where no changes in thyroid hormone levels were seen in humans

receiving doses of triclosan that are in the relevant range encountered from the use of

consumer products.

32




nve stigat ion of otent ial ndocrine ctivity of Triclosan September 24 2 9

As discussed above. Kumar et al. 2009 and Zorrilla et al. 2009 inves tigated the

effects of triclosan on the levels of testosterone , LH and assoc iated critical enzymes in

the production of testosteron e in rats. Due to methodological questions in the Kumar et

al. 2009 study, the results of Zorrilla et al. 2009 are cons idered more re liable to

discern the potential effects of triclosan on these biochemical indices of the potential for

reproductive impacts. The resul ts of Zorrilla et al. 2009 are in agreement with the

experimental data fo r reproductive effects repo rted by Morse th 1988 . As discu ssed in

the Zorril la et al. 2009 study, triclosan did not affect critical parameters , e.g., testes

weight, testoste ron e and LH production or no rmal development of puberty at

concentrations up to the highest dose tested, 300 mg/kg/day, which is higher than the

highest dose tested in the generation reproductio n study conducted by Morseth 1988 .While the in vitro studies provid e insights into the potentia l ways in which tric losan could

affect various enzymes involved in testos terone biosyn thes is, these studies are only useful

as screens to gene rate hypo theses for such effects not as definitive evidence of endocrine

disruption leading to reproductive tox ici ty. In light of the classic negativ e

reproduc tive/developm en tal toxicity tests, the changes noted in these in vitro tests are of

interest and may explain chang es at high triclosan con cen trat ions when normal

phy siological processes , e.g., feed back loops, are no t operative but are not re levant to or

predictive of human health outcomes at envi ronmentally re levan t exposures.

It is important tha t these results be put in pe rspect ive, not only as they compare to

the results from a ba ttery of class ic rep rod uct ive/developmental studies that have been

conducted in exper imental an imals, bu t also as these exposures compare to those

anticipated for users of consumer products con tain ing triclosan. A large biomonitoring

study, which includes triclosan has been conduc ted by the CDC as part of the NHANES

survey, which has been conducted annually since 1976. The NHANES study was

designed to measure the hea lth and nu trit ional status in the general US population

Calafat al. 2008 . Recently, the survey has included data from urine and plasma

samples to assess exposure to environmental chemicals.

In the 2003 -2004 NHANES survey CDC 2005 , which included exam inations of

9.643 subjects, urine specimens were co llec ted from a random one-third of the subjects

n=2,5 17 subjects and analyzed fo r tric losan Calafat et al. 2008 . A sing le spot u rine

33





sample was collected from each subject during one of three daily examination sessions,

either morning, afternoon or evening. The samples were analyzed for concentrations of

free plus conjugated triclosan total triclosan) in the urine and adjusted for volume or

total creatinine. These data provided actual measurements of triclosan from a large

number of subjects of various ages that potentially used multiple triclosan containing

products. Because of the pharmacokinetic properties of triclosan in the human i.e.,

approximately 90 urinary excretion of parent and metabolites and no bioaccumulation),

the urinary biomonitoring data provided by the NHANES survey DC 2005; Calafat et

al. 2008) can be used to provide estimates of the absorbed daily intake of triclosan

resulting from exposure to triclosan, irrespective of source. A similar approach has been

used to estimate pesticide exposure Mage Ct al. 2004). f it is assumed tha t the individuals have a relatively constant use of triclosan

products resulting in a constant daily intake, the samples taken as part of the NHANES

survey would represent a steady state concentration of triclosan in the urine. These

urinary concentrations can be adjusted by creatinine and body weight the subject to

estimate a daily intake in units of mg/kg/day. The resulting daily triclosan intake

estimates based on the 50th percentile, assumed to represent the average user, urinary

concentrations of triclosan reported in the NHANES 2003-2004 CDC 2005) survey were

approximately 0.0002, 0.0002 and 0.0001 mg/kg/day for men, women, and children,

respectively Rodricks et al. 2009).

Based on these estimates, the highest estimated daily intake of triclosan from

consumer products would be approximately 0.2 jig/kg/day, which is more than ten-

thousand-fold lower that the No Observed Adverse Effect Levels NOAELs) in

experimental animals based on changes in hormone levels, which was mg/kg/day for

the lack of changes in thyroid hormone levels or 300 mg kg day for the lack of effects on

hormones in the reproductive system, both of which were reported by Zorrilla et al.

2009). While some changes in hormone levels associated with thyroid function or with

male reproductive function in rats have been reported, it is unlikely that triclosan will

have similar effects in humans because: humans have considerable redundancy and

robust mechanisms to maintain homeostatic functioning; and 2) expected exposures to

and resulting average daily intakes from the use of consumer products containing

34




Investigation of otential Endocrme ctivity of Triclosan September 24 9

triclosan are orders of magnitude below the levels at which changes in hormone levels

and other indices of endocrine action have been reported in animals Therefore the

recent studies in rats and in vitro systems that evaluated the potential effect of triclosan

on the endocrine system specifically endocrine function as it relates to thyroid function

and reproductive function are unlikely to be relevant to humans and so do not pose a

safety concern for triclosan use in humans

35




nvestigation of Potential ndocrine ctivity of Triclosan eptember 24 2 9

5.0 REFERENCES

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nvestigation of otential ndocrine Activity of riclosan September 24 2 9

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Kumar, V., Balomajumder, C., and Roy, P 2008 . Disruption of LH-inducedtestos terone biosyn thes is in testicular Leydig cells by triclosan: probablemechanism of action. Toxicology. 250 2-3 124-131.

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Leuschner, F Leuschner, A., Schwerdtfeger, W., and Dontenwill, W 1970 . 90 DaysOral Toxicity Study in Beagle Dogs with CH 3565. Labortorium furPharmako logie und Toxiko log ie. July 10

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Investigation of Potential ndocrine ctivity of riclos n September 24 2 9

Molitor, E., Persohn , E and Thomas, H 1992). The effect of fat 80’023 /Q I rgasan DP300) on selected biochemical and morpho log ical liver parameters fol lowingsubchron ic dietary administ ra tion to male and female mice. Ciba-Geigy Limited.Laboratory Repo rt No. CB 91/18.

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nv estigat ion of Potentia l ndocrine c tiv ity of Tric los an eptember 24 2 9

Saghir, S., Charles, G., Bartels, M., Kan, L., Dryzga, M., Brzak, K., and Clark, A. 2008 .Mechanism of triflura lin induced thyroid tumors in rats. Toxicology Letters.180 1 38-45.

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Investigation of otenti l ndocrine ctivity of Tric losan September 24 2 9

Wang, IQ., Falany CN., James MO. 2004 . Triclosan as a substrate and inh ibitor of 3’-phosphoadeno sine 5’ phosphosulfa te su lfotransgerase and UDP-glucuronosy ltransferase in human liver fractions. Drug Metabol ism Dispo. 32:1162-1169.

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SCCP/1192/08

Scientific Committee on Consumer Products

SCCP

OPINION ON

Triclosan

COLIPA n° P32

The SCCP adopted this opinion at its 19th plenary of 21 January 2009



SCCP/1192/08

Opinion on triclosan

About the Scientific CommitteesThree independent non-food Scientific Committees provide the Commission with thescientific advice it needs when preparing policy and proposals relating to consumer safety,public health and the environment. The Committees also draw the Commission's attention

to the new or emerging problems which may pose an actual or potential threat.

They are: the Scientific Committee on Consumer Products (SCCP), the Scientific Committeeon Health and Environmental Risks (SCHER) and the Scientific Committee on Emerging andNewly Identified Health Risks (SCENIHR) and are made up of external experts.

In addition, the Commission relies upon the work of the European Food Safety Authority(EFSA), the European Medicines Evaluation Agency (EMEA), the European Centre forDisease prevention and Control (ECDC) and the European Chemicals Agency (ECHA).

SCCPQuestions concerning the safety of consumer products (non-food products intended for theconsumer).In particular, the Committee addresses questions related to the safety and allergenicproperties of cosmetic products and ingredients with respect to their impact on consumerhealth, toys, textiles, clothing, personal care products, domestic products such asdetergents and consumer services such as tattooing.

Scientific Committee membersClaire Chambers, Gisela Degen, Ruta Dubakiene, Bozena Jazwiec-Kanyion, VassiliosKapoulas, Jean Krutmann, Carola Lidén, Jean-Paul Marty, Thomas Platzek, Suresh ChandraRastogi, Jean Revuz, Vera Rogiers, Tore Sanner, Günter Speit, Jacqueline Van Engelen, IanR. White

ContactEuropean CommissionHealth & Consumer Protection DGDirectorate C: Public Health and Risk AssessmentUnit C7 - Risk AssessmentOffice: B232 B-1049 [email protected]

© European Commission 2009

(ISSN)

The opinions of the Scientific Committees present the views of the independent scientistswho are members of the committees. They do not necessarily reflect the views of theEuropean Commission. The opinions are published by the European Commission in theiroriginal language only.

http://ec.europa.eu/health/ph_risk/risk_en.htm



SCCP/1192/08


ACKNOWLEDGMENTS

Dr. C. ChambersProf. G. Degen (rapporteur)Dr. B. Jazwiec-KanyionProf. V. KapoulasProf. J.-P. MartyProf. T. PlatzekDr. S.C. RastogiProf. J. RevuzProf. V. RogiersProf. T. Sanner (chairman)Dr. J. van EngelenDr. I.R. White

Keywords: SCCP, scientific opinion, preservative, triclosan, P32, directive 76/768/ECC,CAS 3380-34-5, EINECS 222-182-2

Opinion to be cited as: SCCP (Scientific Committee on Consumer Products), Opinion ontriclosan, 21 January 2009



SCCP/1192/08


TABLE OF CONTENTS

ACKNOWLEDGMENTS ………………………………………………………………………………... 3

1. BACKGROUND …………………………………………………………………………………. 5

2. TERMS OF REFERENCE …………………………………………………………………………………. 5

3. OPINION …………………………………………………………………………………. 6

4. CONCLUSION ………………………………………………………………………………….

123

5. MINORITY OPINION ………………………………………………………………………………….

123

6. REFERENCES ………………………………………………………………………………….

123



SCCP/1192/08


1. BACKGROUND

Triclosan (CAS 3380-34-5) with the chemical name 5-chloro-2-(2,4-dichlorophenoxy)phenolor 2,4,4'-trichloro-2'-hydroxy-diphenyl ether has a long history of use as a preservative in

cosmetic products. It is currently regulated in Annex VI, entry 25 with a maximumconcentration of 0.3%.

An opinion on triclosan (SCCP/1040/06) was adopted by the SCCP at the 9th plenarymeeting of 10 October 2006 with the following conclusions to the request:

1. "On the basis of the available data, the SCCP is of the opinion that there is presentlyno evidence of clinical resistance and cross-resistance occurring from the use oftriclosan in cosmetic products. Information is required on consumer exposure totriclosan from all sources, including cosmetic products.

2. For a toxicological assessment of the safe use of triclosan, the SCCP requires a dossier

to be submitted in which data is provided to all relevant exposure and toxicologicalend-points and conforming to currently accepted standards. This should be regardedas a matter of urgency because triclosan has been identified in human milk of someEuropean populations."

The dossier provided by Industry consists of an update on the bacterial resistance issue(submission III) and of a toxicological dossier for triclosan (submission IV).

Furthermore the Norwegian authority on cosmetics has earlier this year submitted a report"Risk assessment on the use of triclosan in cosmetics; Development of antimicrobialresistance in bacteria – II".

2. TERMS OF REFERENCE

1. Does SCCP consider a continued use of triclosan as a preservative in cosmetic products as safe for the consumer at the current concentration limit of maximum0.3% taking into account the provided toxicological data?

2. Does SCCP consider a continued use of triclosan as a preservative in cosmetic

products as safe taking into account the new provided documentation of resistancedevelopment by certain micro-organisms and cross-resistance?



SCCP/1192/08


3. OPINION

This opinion only addresses possible toxicological effects of triclosan on human health(question 1 in Terms of Reference). It does not cover aspects of potential resistance

induction in micro-organisms by triclosan, which will be treated in a separate opinion.

3.1. Chemical and Physical Specifications

3.1.1. Chemical identity

3.1.1.1. Primary name and/or INCI name

Triclosan (INCI)

3.1.1.2. Chemical names

2,4,4’-trichloro-2’-hydroxy-diphenylether

3.1.1.3. Trade names and abbreviations

Irgasan® DP300, Irgasan® PG60, Irgacare® MP, Irgacare® CF100, Irgacide® LP10

Triclosan is also referred to as Irgasan, DP300, FAT 80’023), CH 3565, and GP 41-353 in anumber of toxicology studies

3.1.1.4. CAS / EINECS number

CAS: 3380-34-5EINECS: 222-182-2

3.1.1.5. Structural formula

O

CI

OHCI

CI

3.1.1.6. Empirical formula

Formula: C12H7Cl3O2

3.1.2. Physical form

White crystalline powder

3.1.3. Molecular weight

Molecular weight: 289.5



SCCP/1192/08


3.1.4. Purity, composition and substance codes

Identification: IR Spectroscopy

The purity of batches of triclosan used in personal care products since the 1970s has beenas described in the following table:

Purity Specifications for triclosan since 1970

Test Point Effectivefrom 1970

Effectivefrom

26.09.1985

Effectivefrom

1.1.1990

Effectivefrom

31.12.1994

Effectivefrom

26.6.2000

Effectivefrom

06.11.2003

TriclosanActiveSubstance1

99.0 -100.0%

99% min 99% min 99.0-100% 97.0-103.0% 97.0 -103.0%

1 Analysis by gas chromatography

The purity of different batches of triclosan used in the toxicology studies is described in thefollowing table.

Purity of triclosan Batches Used in Toxicology Studies

Triclosan Batch Number/Information Purity1

FAT 80’023/AMischung 652

99.3%

FAT 80’023/BCH3565, Mg 120

99.3

FAT 80’023P4-11-210 , Package Nr. RP68118

99.9

FAT 80'023 BAIrgasan DP300, Batch #5.2.0211.0

99

Lot No. S 15155 T01 = Unilever No. S15155 T01 ≥99%FAT 80’023/H5/0/0194/0, Batch No. EN46856.02

Within specifications2

FAT 80’023/QBatch No. EN 91390.76

99.6

Triclosan R&D name: GP41353PBS 5357.0, Lot No. 800187

100.3

C-P sample No 39317Lot 19851206, Irgacare MP

101

C-P sample No. 38328Lot 19851206, Irgacare MP

101

FAT 80’023/RBatch No. EN 275927.26

99.5%

FAT 80’023/SBatch No. 505017

99.5%

Lot 020750A7 99.8

P&G No. D1063.01D1063.02R

99.7

1 Analysis using gas chromatography2 A compiled analytical report is not available, but data shows the batch was within specifications.

Water content: ≤ 0.1%



SCCP/1192/08


3.1.5. Impurities / accompanying contaminants

Individual related compound (GasChromatography)

≤0.1%

Total related compounds (Gas Chromatography) ≤ 0.5%2,4 Dichlorophenol ≤10 mg/kg

Sum of 3- and 4-Chlorophenol ≤10 mg/kg

2,3,7,8 Tetrachlorodibenzo-p-dioxin <0.001 µg/kg

2,3,7,8-Tetrachlorodibenzo-furan <0.001 µg/kg

2,8-Dichloroldibenzo-p-dioxin ≤0.5 mg/kg

1,3,7-Trichlorodibenzo-p-dioxin ≤0.25 mg/kg

2,8-Dichlorodibenzo-furan ≤0.25 mg/kg

2,4,8-Trichlorodibenzo-furan ≤0.5 mg/kg

Ash ≤0.1%

Mercury ≤1 mg/kg

Arsenic ≤2 mg/kg

Antimony 10 mg/kg

Lead ≤10 mg/kg

Cadmium ≤5 mg/kg

Nickel ≤10 mg/kg

Copper ≤10 mg/kg

Chromium ≤2 mg/kg

Sum of heavy metals as lead sulfide precipitation ≤20 mg/kg

3.1.6. Solubility

Solubility of triclosan in some selected solvents and chemicals

Solvent Solubility at 25°C (g triclosan/100 g solvent)

Distilled water (20°C) 0.001


1 N caustic soda 31.7

1 N sodium carbonate 0.40

1 N ammonium hydroxide 0.30

Triethanolamine >100

Acetone >100

Ethanol 70% or 95% >100

Isopropanol >100

Propylene glycol >100

Polyethylene glycol >100

Methyl cellosolve (Union Carbide Corp.) >100

Ethyl cellosolve (Union Carbide Corp.) >100

Dipropylene glycol ~40

Glycerine 0.15

n-Hexane 8.5

Petroleum jelly (white, USP) ~0.5

Tween 20 (ICI America Inc.) >100



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Solubility of triclosan in some selected solvents and chemicals

Solvent Solubility at 25°C (g triclosan/100 g solvent)


Triton X-100 (Rohm & Haas) >100

Olive oil ~60

Castor oil ~90

3.1.7. Partition coefficient (Log Pow)

Log Po/w: 4.8

3.1.8. Additional physical and chemical specifications

Melting point: 57 ± 1°CBoiling point: /Relative density: 1.55 ± 0.04 g/cm3

Vapour pressure: 4 x 10-6 mmHg (20°C)Viscosity: /pKa: 8.14 (20°C)Refractive index:UV_Vis spectrum (200-800 nm): /

3.1.9. Homogeneity and Stability

The stability of triclosan under normal storage conditions (ambient temperature) has beenassessed with a batch produced in 1973 and re-analyzed 4 and 9 years after manufacturing.The study showed that triclosan does not decompose under normal storage conditions; thequality has remained constant over the 9 years of storage.

Storage time Starting time (1973) After 4 years (1977) After 9 years (1982)

Appearance White, fine crystalline White, fine crystalline White, fine crystalline

Content activesubstance

99% 99.7% 99.5%

1 Irgasan Dp 300, Batch No. EN 30142 storage conditions: ambient temperature

General Comments to physico-chemical characterisation

- Stability of triclosan in marketed products is not reported.

3.2. Function and uses

Triclosan is an antibacterial ingredient that has been used in consumer products for over 30years. It is widely used as a non-ionic antibacterial agent in personal care products (e.g.,bar and liquid soaps, deodorants (Danish EPA, AR5), skin-care products, foot-care productsoral care products, and make-up products). Triclosan was approved in 1986 by theEuropean Community Cosmetic Directive for preservative in cosmetics products atconcentrations up to 0.3%. Triclosan was evaluated also by SCF [SCF, 2000, AR8] and EFSA[EFSA, 2004, AR6] for use in food contact materials and classified in SCF_List 3 with arestriction of 5 mg/kg of food.



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Other fields of applications are textiles (i.e., sport wear) and plastic materials (i.e., plasticcontainers, brushes), with a triclosan concentration of up to 0.3%.In the EU, about 85% of the total volume of triclosan is used in personal care products,compared to 5% for textiles and 10% for plastics and food contact materials. The quantityused within the EC reached approximately 450 tons (as 100% active) in the year 2006.At or near typical usage levels, triclosan appears to intercalate into the membrane of

bacteria, resulting in destabilised structure/function. This level of exposure results indisruption of nutrient uptake, inhibition of amino acid incorporation, inhibition of uracilincorporation, as well as membrane lysis.

3.3. Toxicological Evaluation

A number of the animal toxicology studies were conducted prior to the publication of GLPstandards and the establishment of OECD guidelines for the conduct of such studies; ofthese studies, many were still considered relevant, based on comparability to OECDguidelines. However, other non-GLP/non-OECD animal studies provided limited supportiveinformation. The pivotal repeated-dose, sub-chronic and chronic studies were conducted

pursuant to GLP regulations and generally followed the OECD guidelines. These pivotalstudies are described in more detail.

3.3.1. Acute toxicity

A number of acute toxicity studies have been conducted for triclosan using the oral, dermal,intraperitoneal or intravenous routes of administration in mice, rats, rabbits, and dogs. Thestudy designs were not always consistent with OECD guidelines for acute toxicity studiesand there were no GLP compliance statements. A number of the acute toxicity studies wereconducted prior to the establishment of GLP regulations.

3.3.1.1. Acute oral toxicity

Triclosan is not acutely toxic via the oral route of administration, with high oral intubationLD50 values in the range of 3,750 to 5,000 mg/kg bw in mice and rats, and an oral capsuleLD50 value of greater than 5,000 mg/kg bw in dogs.

Ref.: 1

3.3.1.2. Acute dermal toxicity

The dermal LD50 value for triclosan was reported to be greater than the high dose of9,300 mg/kg bw tested in rabbits. These data indicate that triclosan is not acutely toxic via the dermal route of administration.

Ref.: 1

3.3.1.3. Acute inhalation toxicity

No acute inhalation toxicity studies were available.

3.3.1.4. Additional acute toxicity studies

Mice and rats administered triclosan intravenously at 10, 20 and 30 (rats only) mg/kg bw ina vehicle solution of triethyleneglycol/water (1/2) showed signs of slight cramps,exopthalmos (mice only), mydriasis (rats only), dyspnoea, and ventral decubitus, withrecovery by 24 to 48 h after dosing.

Ref.: 2, 3



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Mice in the intraperitoneal studies showed signs of general lethargy, lassitude, closehuddling, lack of response to tactile stimuli and no increase in respiration rate andhyperactivity with death occurring between 6 and 72 hours.

Ref.: 4, 5

Data from the study by Kanetoshi et al. (1992) were also mentioned in a review by

Bhargava and Leonard (1996).Additional References Bhargava, Leonard 1996

Table 1: summary of LD50 values from toxicity studies in mice, rats, rabbits, and dogs

Species Route of Administration LD50 (mg/kg) Reference;GLP and OECD Status

Mouse (NMRI) Intravenous 19 Walther, 1968a (2);

Predates GLP and OECD

Mouse Oral Gavage 4,350 DeSalva et al., 1989 (1);

Not reported, but some studieslikely predate GLP and OECD

Mouse (CD-1) (Female) Intraperitoneal 184 Miller et al ., 1982 (4);

GLP: not reportedOECD: no comparable guidelines

Mouse (ddy) (Male) Intraperitoneal 1,090 Kanetoshi et al., 1992 (5);

GLP: not reportedOECD: no comparable guidelines

Rat (Wister CFE) Intravenous 29 Walther, 1968b (3);

Predates GLP and OECD

Rat Oral Gavage 3,750 to 5,000 DeSalva et al., 1989 (1);


Rabbit Dermal >9,300 DeSalva et al., 1989 (1);


Dog Oral Capsule >5,000 DeSalva et al., 1989 (1);




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3.3.2 Irritation and corrosivity

3.3.2.1. Skin Irritation

Table 2: Findings from Non-GLP Skin Irritation Studies with triclosan

Species

(Strain)

Application Details Major Findings Reference,

GLP andOECD

Status

Guinea pig(Pirbrightwhite)

0.1 mL single application,test site: 2 cm2 area on theshaven back, concentrationsof 0.1, 0.5, 1.0, and 5.0%(duration of exposure andocclusion status unknown)

Erythema reactions were only observed in thehighest dose group (4/10 positive responses) at 24hours post-application. No positive responses werereported at 48 hours.

Thomannand Maurer,1978(7)

Predates GLPand OECD

Rabbit

(Russian)

Triclosan-soaked 2.5 cm2

gauze patches with occlusivedressings applied to shavedintact skin or abraded skinsites on the backs and flanksof the rabbits for 24 h(concentration of triclosanunknown).

Erythema reactions were observed at 24 h after the

start of exposure [3/6 positive responses in intactskin (mean score: 2.5); 5/6 positive in abraded skin(mean score 2.8)]. Oedema reaction at 24 h waspositive in 1/6 animals. Erythema reactionsimproved at 72 h (mean scores: 1.3 and 2.5 in intactand abraded skin, respectively). Triclosan did notinduce corrosion effects. Triclosan was classified as amoderate irritant based on an overall score of 3.58(irritance scores of greater than 6 would have beenconsidered “severe”).

Sachsse and

Ullmann,1975(6)


The results of the guinea pig study suggests that triclosan is not a skin irritant atconcentrations below 5%, while both studies show that the erythema reactions arereversible.

Skin irritation data were also available from 14-day repeated-dose and 90-day sub-chronicdermal toxicity studies of triclosan in the rat, mouse, and newborn monkey (Tables 10, 14and 15). In the 14-day mouse and rat studies, conducted as dose range finding studies forlonger-term repeated dose toxicology studies [Burns, 1997a (8); Burns, 1996 (9); Burns,1997b (10)], erythema and scaling were observed in the mouse at doses of1.5 mg/animal/day and higher (applied as solutions of 1.5 to 6%). Erythema, oedema,fissuring, eschar, alopecia, thickening and discoloration of the test site were noted in the ratat doses of 3.0 and 6.0 mg/animal/day (applied as solutions of 1% and 2% in 0.3 mLacetone). These studies show that repeated topical application of triclosan (up to 6%)resulted in moderate to severe skin irritation in the rat and mouse. In the 90-day sub-chronic toxicity study in rats, reversible skin irritation was observed starting at the lowest

dose tested of 10 mg/kg bw/day (a concentration of approximately 0.5% triclosan inpropylene glycol applied in a volume of 2 mL/kg body weight) [Trimmer, 1994 (11)]. In anearly non-GLP study, dermatitis was observed in weanling dogs treated for 90 days with200 mg/kg body weight/day, but not in dogs treated with the lower doses of 20 or 2 mg/kgbw/day [Dorner, 1973 (12)]. Limited and selective reporting of findings made theinterpretation of the data from this dog study unreliable. In an early non-GLP study innewborn Rhesus monkeys bathed using a 15 mL of a 0.1% soap solution containingtriclosan, no signs of dermal irritation were observed after 90 days of daily bathing[Hazleton Laboratories, 1979a (13)].

Additional skin irritation data are available from studies in humans (Section 3.3.11.3-1).



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3.3.2.2. Mucous membrane Irritation

Table 3: Findings from a Mucosal Irritation Study with Triclosan in Hamsters

Species(Strain) Application Details Major Findings Reference;GLP and

OECD Status

Hamster(Syriangolden)

1.5% sodium lauryl sulphate(SLS), 1.5% SLS and 0.3%triclosan, or 0.3% triclosan inpaste, 1 cm2 area of the rightcheek pouch, 4 applications for15 seconds at 24-hour intervals.

The histological structure of the mucosa of thetriclosan-treated animals (Group 3) was similarto that of the control specimens. Structuralchanges, including basal hyperplasia,acanthosis, hypergranulosis, and orthokeratotichyperkeratosis, were present in animals treatedwith SLS, or with SLS and triclosan. No signs ofinflammation were observed in the subepithelialconnective tissue.

Baert et al .,1996(14);GLP: notspecified

The results of this study show that the application of triclosan in a paste (0.3% triclosan)

does not result in mucosal irritation in the hamster cheek pouch.

Table 4: Findings from a GLP Eye Irritation Study with Triclosan

Species

(Strain)

Application Details Major Findings Reference,

GLP andOECD Status

Rabbit(NewZealandWhite)

0.1 g of pure solid triclosan wasinstilled into the conjunctival sacof the left eye (control: righteye). Examinations were onDays 1, 2, 3, 4, and 7.

Based on mean irritation scores in the cornea,iris, and conjunctiva, triclosan was found tocause slight primary eye irritation when appliedto the rabbit eye mucosa.

Ullmann,1980 (15);

GLP:comparable

OECD 405consistent

Table 5: Findings from a Non-GLP Eye Irritation Study with Triclosan

Species

(Strain)

Application Details Major Findings Reference;

GLP andOECD Status

Rabbit(strain notreported)

Triclosan was applied either ingum Arabic (1, 2, 5, 10, or 20%concentrations) into the

conjunctival folds (observationsup to 24 h), or as an undilutedsubstance (observations up to11 days) without rinsing, or withrinsing after 2 or 4 seconds ofexposure.

Triclosan at concentrations of 1, 2, 5, and 10%produced only slight to moderate reddening ofthe conjunctiva as observed 2 hours after

exposure, with recovery within 24 hours.Triclosan at a 20% concentration causedreddening and slight swelling of the conjunctivaas observed 24 hours after the start ofexposure. The undiluted (pure) triclosan causedfully reversible eye irritation effects, asobserved in rinse experiments, with a return tonormal eye state between 7 and 11 days afterexposure.

Lyman andFuria, 1969(16);


The results of eye irritation studies in rabbits showed that triclosan causes slight eyeirritation when tested in its undiluted form. Triclosan did not produce either severe irritationor corrosive effects. Minimal to slight irritation effects to the cornea, iris, and conjunctivaewere observed in tests using pure triclosan, resulting in an overall irritation score thatindicated slight primary eye irritation [Ullmann, 1980 (15)]. Similarly, when tested atconcentrations of 1, 2, 5, and 10% in gum Arabic, triclosan was shown to cause only



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reddening of the conjunctivae as observed at 2 hours, which was fully reversed within 24hours after the start of exposure [Lyman and Furia, 1969 (16)]. Triclosan at the highconcentration of 20% caused slight to pronounced reddening and slight swelling of theconjunctiva that was still observed after 24 hours.

Summary of Irritation/Corrosivity Data

Triclosan was a skin irritant at a level of 5% but not at 1% in the guinea pig skin irritationstudy. Repeated topical application of 1 to 6% triclosan for 14 days in dermal toxicity doserange-finding studies resulted in moderate to severe skin irritation in the rat and mouse.Lower concentrations were without effect in these studies. However, all of the doses in the14- and 90-day dermal toxicity studies are considerably higher than exposures expectedfrom the use of triclosan-containing personal care products. Effects were also observed in askin toxicity study of 90 days duration in the rat at a concentration of 0.5%. In summary,the irritation/corrosivity data from either irritation studies in the hamster, guinea pig, andrabbit, or skin toxicity studies conducted in the mouse, rat, monkey, and dog suggest thattriclosan may cause slight reversible skin irritation at concentrations of 0.5 to 5% underexperimental conditions.

Triclosan was not an irritant to mucous membranes in the hamster cheek pouch assay at alevel of 0.3%.Triclosan at concentrations of 1 to 10% produced only slight, reversible irritation in therabbit eye.

3.3.3. Skin sensitisation

Table 6: Findings from Sensitisation Studies with Triclosan

Species

(Strain)

Application Details Major Findings Reference; GLP

and OECD

Status

Guineapig,Hartley(albino)

Guinea pig Buehler test.N=5 treated animals, 6control animals. 1%triclosan in a cream/gel(occluded dermalapplication for bothinduction and challenge).Induction: 9 5-hourexposures on backs ofanimals. No adjuvant.Challenge: 14 to 21 daysafter induction.

Induction: Slight primary irritation was observedafter the first few treatments but was alleviatedwith regular wash-off procedures.Challenge: Treated sites showed slight irritation(redness) 24 and 48 hours after the challenge.Previously untreated sites did not show anysignificant oedema or erythema after challenge.Skin contact sensitization did not occur.

ToxicologicalResources, 1974(17);


Guineapig,Hartley

(albino)

Split Adjuvant method.N=20 animals/treated orcontrol group. Induction:

triclosan (10% in petrola-tum) applied 3 times.Complete Freund's adjuvantadministered intradermallybetween 2nd and 3rd induction doses. Challenge:triclosan (3% inpetrolatum) applied once 13days after induction.

Induction: Slight erythema without oedema orvesiculation was observed in 7 of 20 triclosan-treated animals. Erythema disappeared 1 or 2

days after the last application. Challenge: Brightpink and moderately elevated reaction in 1 of 20animals at 24 and 48 hours post-challenge. At 72hours, erythema was still present but withoutoedema. There were no reactions in any of thecontrol group animals. The authors concludedthat triclosan has a very low sensitisation index.

Lachapelle andTennstedt, 1979(18);




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Species(Strain)

Application Details Major Findings Reference; GLPand OECD

Status

Guineapig,Pirbrightwhite

Modified Maximisation test(Optimization).N=10/sex/group.Induction: 0.1% triclosan inpropylene glycol, 6 intracu-taneous injections over 3weeks. (Complete Freund'sadjuvant was used duringthe 2nd and 3rd weeks.) 1st challenge: 14 days afterlast induction,intracutaneous (0.1%triclosan in 40% propyleneglycol). 2nd challenge: 14days after 1st induction,occluded patch (0.1%triclosan in petrolatum).

Induction: Erythema results were not reported forthe induction phase.Challenge: The incidence of positive reactions wassimilar for triclosan-treated and control animals(4/20 after first challenge compared to 4/19 in thevehicle control group; 3/20 after second challengecompared to 1/19 in the vehicle control group).The investigators concluded that triclosan showedno skin-sensitising potential in this study.

Maurer et al .,1979(19);


Guinea pig

(Hartleyalbino)

Guinea pig Buehler test.

N=10/group. Induction:25% triclosan initially(reduced to 10%, then 2%(topically applied 3 times /week for 3 weeks). Noadjuvant. Challenge: 5%triclosan in propyleneglycol, considered thehighest non-irritatingconcentration, applied to anaïve site. Positive control:DNCB treatment.

GLP-compliant. Conducted according to OECD

Guideline No. 406. Skin reactions scoring >0.5were considered to be positive (maximum score:3).Induction: very faint to severe erythema,depending on the dose level (note that doses werecontinually reduced to reduce irritation).Challenge: After challenge, triclosan treatmentinduced weak, non-confluent reactions of veryweak erythema (scores of 0.5) in 6/10 animals(not considered to indicate sensitisation). Therewere no scores greater than 0.5 among triclosan-treated animals. Negative controls showed similarfaint erythema reactions in 2/5 animals (scores of0.5). Positive controls showed strong reactions(scores of 1 to 3) in 10/10 animals (meanerythema scores of 1.8). The investigatorsconcluded that triclosan is not a contactsensitizer.

Wnorowski,

1994(20);

GLP: compliant

OECD: No.406consistent

Triclosan as tested in 4 studies was found not to cause skin sensitisation. Both the 1974and the 1978 studies used methods that were similar to those recommended by currentOECD guidelines for sensitisation studies, and the more recent 1994 study was GLP-compliant and followed OECD (No. 406).

In a small Buehler test in guinea pigs, slight irritation was observed during the inductionperiod with 1% triclosan, attributed to build-up of the cream formulation on the test site[Toxicological Resources, 1974 (17)]. The study investigators concluded that nosensitisation was observed, as previously untreated sites did not show any significant

oedema or redness following the challenge dose. Slight irritation (erythema withoutoedema) also was observed during induction in a much larger sensitisation study using the

“split adjuvant” method in guinea pigs [Lachapelle and Tennstedt, 1979 (18)]. In thisstudy, a positive sensitisation reaction following challenge was observed in only 1 of 20animals. Thus, these authors also concluded that triclosan has a very low sensitisationindex. In the third study, a modified maximisation test in guinea pigs, there was nosignificant difference in sensitisation reactions following challenge in animals treated with0.1% triclosan in propylene glycol compared to animals treated with the vehicle alone[Maurer et al ., 1979 (19)]. In the GLP study, a Buehler assay conducted following OECDguidelines, triclosan showed no evidence of sensitisation potential, in contrast to thepositive control substance [Wnorowski, 1994 (20)]. Altogether, the results of these studiessuggest that triclosan is not a sensitising agent as tested in the guinea pig.



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Summary of Sensitisation Data

Triclosan has been tested in skin sensitisation studies in guinea pigs, with both adjuvantand non-adjuvant methods used.In summary, the results of these GLP and non-GLP studies indicate that there is no evidence

for sensitisation with triclosan in various formulations and concentrations (up to 10% inpetrolatum) in the guinea pig.

Results of related studies in Humans are reported in section 3.3.11.

3.3.4. Dermal / percutaneous absorption

I n V it r o Studies of Dermal/Percutaneous Absorption

A single in vitro percutaneous absorption study with triclosan was conducted in rat skin[Moss et al ., 2000 (21)]. The main findings in this study are provided in Table 7. After 24hours, 58% of the applied dose remained on the skin surface and in the stratum corneum

(33 and 25%, respectively) and 41.2% of the applied dose was recovered in the receptorfluid and in the epidermis and dermis (23 and 18.2%, respectively). Thus, it can beconsidered that 41.2% of the applied dose was absorbed percutaneously within 24 hours(penetration through the stratum corneum into deeper layers of the skin is considered torepresent absorption). Triclosan was primarily absorbed through the skin as the parentcompound, with some glucuronide and sulphate conjugates being detected in the receptorfluid. Glucuronidation was the primary route of metabolism of triclosan in rat skin.

Table 7: Findings from an In Vitro Percutaneous Absorption Study for Triclosan in Rat Skin

Method Major Findings Reference;GLP Status

Diffusion cell system usingdorsal skin. Seven µL of64.5 mM [3H]-triclosan in anethanol-water (9:1, v/v)solution (0.48 Mbq) wasapplied to the exposed skinsurface (0.64 cm2).

After 24 hours, approximately 23% of the applied dose appearedin the receptor fluid, and 33%, 25%, and 18.2% remained on theskin surface, stratum corneum, and epidermis and dermis,respectively. Of the radioactivity in the receptor fluid at 24hours, 17.3% of the applied dose was present as triclosan, 4.1%as triclosan glucuronide, and 0.9% as triclosan sulfate. Of theradioactivity in the skin at 24 hours, 13% of the dose wasrecovered as triclosan, 1.4% as triclosan glucuronide, and 1.6%as triclosan sulfate.

Moss et al., 2000 (21);

GLP: notspecified

I n V iv o Studies of Dermal/Percutaneous Absorption

The main findings in the in vivo percutaneous absorption studies for triclosan are providedin Table 8.

Table 8: Findings from In Vivo Percutaneous Absorption Studies for Triclosan

Species Method Major Findings Reference;

GLP Status

Mouse Single application ofliquid soaps. Test site:1 cm x 3 cm area onthe back. 22.54 to25.49 µg triclosan/cm2 skin.

Triclosan deposition on hairless mouse skin was0.98 ± 0.11 and 1.16 ± 0.13 µg/cm2 skin, followingapplication of approximately 22.54 and 25.49 µgtriclosan/cm2 skin. The percent of the applied dosedeposited on the skin was 4.425 ± 0.617 and 4.809± 1.236, respectively.

Demetrulias,1985 (22);

GLP: notspecified



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Species Method Major Findings Reference;GLP Status

Mouse 0.05 mL of [3H]-Irgasan DP300 (1.6mg, 10 µCi) in olive oilwas applied to a 5 cm2 area of the shavedback.

Maximum tissue levels (at 12 or 18 hours) werehighest in the gallbladder (402 ± 57 µg/g), followedby liver (13.4 ± 3.5 µg/g), fat (10.0 ± 3.5 µg/g),lung (7.5 ± 2.9 µg/g), blood (6.5 ± 1.9 µg/g), andkidney (4.3

± 0.8 µg/g). Maximal levels in the

testes, heart, and spleen were in the range of 1.1to 1.8 µg/g. Low levels were detected in the brain(0.2 to 0.5 µg/g).

Kanetoshi et al.,1992 (5);

GLP: notspecified

Mouse(CD-1)

0, 0.3, 0.6, 1.5, 3.0, or6.0 mg/animal/d inacetone for 14 days,applied to 2x3 cm2 areaon the dorsal area oncedaily.

Toxicokinetic data from a dose range findingrepeated dose dermal irritation study. Two pooledplasma samples per treatment group (5mice/sex/pooled sample). Mean plasma triclosanlevels in males: 32.5, 63.3, 98.8, 148.2, and 173.3µg/mL at doses of 0.3, 0.6, 1.5, 3.0, and 6.0mg/animal/d, respectively. Females: 63.2, 124.8,144.0, 124.0, 295.2 µg/mL for same doses. Notethat absorbed amount as percentage of applieddose was not calculated.

Burns, 1997a(8)

GLP: compliant

Mouse

(CD-1)

0, 0.3, 0.6, 1.5, 3.0, or

6.0 mg/animal/d inpropylene glycol for 14days, applied to 2x3cm2 area on the dorsalarea once daily.

Toxicokinetic data from a dose range finding

repeated dose dermal irritation study. Two pooledplasma samples per treatment group (5mice/sex/pooled sample). Mean plasma triclosanlevels in males: 7.4, 22.2, 38.6, 75.4, and 72.6µg/mL at doses of 0.3, 0.6, 1.5, 3.0, and 6.0mg/animal/d, respectively. Females: 9.2, 33.0,47.0, 101.8, 112.1 µg/mL for same doses. Notethat absorbed amount as percentage of applieddose was not calculated.

Burns, 1996 (9)

GLP: compliant

Rat(Crl:CDBR)

0, 0.3, 0.6, 1.5, 3.0, or6.0 mg/animal/d inacetone for 14 days, ina volume of 300 µL,applied to 2x3 cm2 onthe dorsal area oncedaily.

Toxicokinetic data from a dose range findingrepeated dose dermal irritation study. Plasmasamples were taken from 10 rats/sex/treatmentgroup. Mean plasma triclosan levels in males: 1.0,2.1, 6.6, 14.1, and 31.6 µg/mL at doses of 0.3,0.6, 1.5, 3.0, and 6.0 mg/rat/day, respectively.Females: 1.2, 2.4, 5.2, 9.2, and 18.1 µg/mL. Notethat absorbed amount as percentage of applieddose was not calculated.

Burns, 1997b(10)

GLP: compliant

Rat Triclosan in an ethanolsolution, in shampoo,or in an aerosoldeodorant was appliedto 7.5 cm2 clippeddorsal skin

Ethanol solution: 27.6% of the applied dose wasabsorbed within 48 hours. Blood levels were in therange of 0.07 to 0.30 µg/mL with Tmax at 6 hours.Shampoo: Penetration of [H3]-triclosan wasdependent on concentration and independent ofduration of contact (5, 10, or 20 minutes).Absorption after 48 hours was in the range of 2.8to 4.1% of the applied dose.Deodorant: Report notes difficulty in administeringa standard, accurate, known dose, so refers todata from the application using an ethanol solution

as vehicle.

Black andHowes, 1975(23);

Predates GLP

Rat Triclosan in an ethanol-water (9:1, v/v)solution (6.92 Mbq)was applied to a 9.6cm2 circular area onthe shaved backs ofthe rats.

After 24 hours, 0.88% of radioactivity was in theurine, 11.84% in the faeces, 0.02% in the blood,26.13% on the skin surface, 4.31% in the skin,0.24% in the cage wash, 7.72% in the carcass,36.33% in the stratum corneum, and 1.38% on theskin cover. Recovery of radioactivity was 90.46%of the dose.

Moss et al., 2000(21);

GLP: notspecified

Rat Single application oftriclosan (4 mg/cm2,400 mg/kg, 10 µCi/rat)to the shaven back

The applied dose remained primarily on theadhesive pad (~64%). Absorbed triclosan wasprimarily excreted in the faeces. 0.5%, 14.66%,0.1%, 5.5%, 7.2% of the applied dose wasrecovered in the urine, faeces, blood, skin, andcarcass/tissues, respectively.

Hong et al., 1976(24);

Predates GLP



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Rat Single application oftriclosan solution(ethanol, acetone), incream, or in Vaseline tothe neck

Solutions – within 96 hours, 2.8 to 3.7% and 69 to89% of the applied dose were excreted in the urineand the faeces, respectively. Cream – within 48hours, 1% and 22% of the applied dose wereexcreted in the urine and faeces, respectively.Vaseline – within 144 hours, 13% and 60% of theapplied dose were excreted in the urine and faeces,respectively.

Ciba-Geigy,1976b(25);

Predates GLP

Rabbit Single or repeated (5x)application of triclosanin solution (propyleneglycol, DMSO,nickethamide, orhexane), in cream, orin soap solution (sitenot reported)

Solutions – 47 to 53% and 38% of the applieddose excreted in the urine and faeces, respectively,independent of dose. Creams – 29 to 48% of theapplied dose was excreted in urine, inverselydependent on quantity of cream applied per unitarea. ≤2% excreted in faeces. Soap solutions – 2to 10% of the applied dose was absorbed(radioactivity measured in urine, faeces, skin,expired air, and organs and tissues).

Ciba-Geigy,1976b(25);

Predates GLP

Rabbit Urine-soaked diaperscontaining 6.4 to 26.9

µg triclosan/g wereapplied to intact orabraded skin (dorsaland flank) and re-applied twice at 4-hourintervals, then left onovernight

Trace amounts of triclosan (i.e., lower than thedetection limit) were detected in some of the blood

and faecal samples, but in most cases none waspresent. The amount of triclosan in the urine wasin the range 0.1 to 3.8 µg (no difference betweenanimals with intact or abraded skin).

Ciba-Geigy,1977b

(26);

Predates GLP

Guinea pig Single application orrepeated application(twice daily for 5 days)of soap suspension ornon-soap detergentsuspension was appliedto a 20 cm2 area (40cm2 area in 1experiment) on theclipped skin of the backfor 2 minutes.

Triclosan remained primarily on the stratumcorneum, and small amounts penetrated into theepidermis, dermis, hair follicles, and sebaceousglands. Repeated application did not increaselocalization in any area of the skin. Increasingconcentrations of triclosan resulted in increasingtriclosan deposition throughout the skin depth.Recovery of triclosan in rinse water accounted for80-90% (single application) and 95% (repeatedapplication mean) of the applied activity. Bloodlevels were in the range of 0.002 to 0.006 µg/mLand 0.014 to 0.027 µg/mL after a single application(40 cm2 vs. 20 cm2). The blood level was 0.019µg/mL following repeated applications. Levels oftriclosan in the tissues were extremely low (ng/grange). Triclosan was excreted mainly in the urinewith relatively small amounts in the faeces.

Black et al .,1975(27);

Predates GLP

Dog 1.3 to 5.0 mLmouthrinse, 15minutes daily for 7days

Peak blood levels (6-8 hrs post-dose) were 0.7 to2.7% (mean = 1.4%) of the applied dose. Dailyurinary excretion of free triclosan and conjugateswas in the range of 1 to 4% (mean = 2%) of theapplied dose.

Lin et al ., 1994(28);

GLP: notspecified

Dog Triclosan in water (200mg/kg), once daily for2 weeks, nuchal skin.

Blood levels were 130 and 165 ppb on Day 7 and14, respectively.

Hong et al.,1976(24);

Predates GLP

Monkey 2 Rhesus monkeys, 3days old, were washedonce with a soapsolution containingtriclosan (1 mg/mL).

Blood levels reached a plateau by 8 to 12 hoursand remained up to 24 hours after treatment.Levels of conjugated triclosan (glucuronide andsulfate) in the blood were in the range of 0.25 to0.68 ppm. No free, unconjugated triclosan wasdetected. Triclosan sulfate levels in the bloodincreased, while triclosan glucuronide levelsdecreased with time post-treatment.

Parkes, 1978a(29);

Predates GLP



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Monkey 10 monkeys/groupwere washed daily for90 days with 15 mL ofa soap solution (0.1%triclosan) (size ofapplication area notreported). 5/groupwere retained for a 30-day recovery period.

Levels of conjugated triclosan (glucuronide andsulfate) in the blood ranged from 0.17 to 0.97ppm. No free, unconjugated triclosan wasdetected. Triclosan glucuronide predominated inthe initial samples (Day 1 or 2). Triclosan sulfatepredominated in all subsequent blood samples (80-90% of total present after 90 days). Urinaryconcentrations of triclosan were in the range of 0.3to 4.8 ppm (primarily the glucuronide conjugate).Faecal concentrations of triclosan were in the rangeof <0.1 to 10.5 ppm (primarily the unconjugatedform). Small amounts of triclosan (<0.1 to 1.9ppm) were detected in tissues from treatedanimals. Following the 30-day recovery period,triclosan was detected only in skin samples.

HazletonLaboratories,1979b(30);

Predates GLP

Deposition and absorption of triclosan were investigated in 4 mouse studies. In a skindeposition study, the triclosan residue on mouse skin was approximately 1 µg/cm2 (4.4 to

4.8% of the applied dose) following application of liquid soap containing [

14

C]-triclosan(22.5 to 25.5 µg/cm2) for 10 minutes [Demetrulias, 1985 (22)]. In a percutaneousabsorption study in the mouse, the maximal radioactivity in tissues (observed at 12 or 18hours) following absorption of [3H]-triclosan was in the range of 14 to 67% of the maximaobtained following oral administration (µg/g range) [Kanetoshi et al., 1992 (5)]. Theabsorption of triclosan was assessed in two 14-day repeated dose dermal toxicity studiesthat showed dose-dependent increases in plasma triclosan levels following application oftriclosan in propylene glycol and in acetone vehicles, respectively [Burns, 1996 (9), 1997a(8)]. Triclosan in plasma was detected at the lowest doses used in the studies (0.3mg/mouse/ day, or 12 mg/kg bw/day in a 25 g mouse, giving plasma levels of 7.4 and32.5 µg/mL using propylene glycol and acetone vehicles, respectively). These resultsindicate that triclosan was readily absorbed through the skin and distributed to tissues inthe mouse.

Four percutaneous absorption studies were conducted in the rat. The results of thesestudies show that the extent of percutaneous absorption of triclosan is dependent on thevehicle used for application. The extent of triclosan absorption was in the range of 23 to28% of the applied dose either in ethanol, ethanol/water, soap suspension, or a creamformulation. Greater absorption was observed with triclosan in an aqueous solution or inVaseline, while lower absorption was observed with triclosan in shampoo. These data areshown in Table 9. In addition to the absorption data from these four studies, plasma datafrom rats that received 14 days of repeated dermal applications of triclosan in acetoneshowed dose-dependent increases in plasma triclosan levels starting at the lowest doseused in the study (0.3 mg/rat/day, or 1.2 mg/kg bw/day in a 250 g rat, giving plasma levelsof 1.0 µg/mL) [Burns, 1997b (10)]. Taken together, these data indicate that triclosan is

readily absorbed through the skin of rats.

Table 9: Percutaneous Absorption of Triclosan in the Rat

Vehicle Time Point (hours) % Dose Absorbed Reference

Pure ethanol 48 28 Black and Howes, 1975 (23)

Shampoo 48 3 to 4 Black and Howes, 1975 (23)

Aerosol deodorant1 48 52 Black and Howes, 1975 (23)

Solution (ethanol, acetone) 96 93 Ciba-Geigy, 1976b (25)

Cream 48 23 Ciba-Geigy, 1976b (25)

Vaseline 144 73 Ciba-Geigy, 1976b (25)

Ethanol/water (9:1) 24 21 Moss et al., 2000 (21)



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Soap suspension 72 28 Hong et al., 1976 (24)1 The determination of absorption following aerosol application was reported to be difficult by the authors of thisstudy due to the lack of a standardized application of the spray. As such, the value of 52% should be interpretedcautiously.

The percutaneous absorption of triclosan was also investigated in the guinea pig [Black et

al ., 1975 (27)]. Autoradiography of skin after a single application of a soap suspensioncontaining radiolabelled triclosan ([3H] or [14C]) showed that triclosan was absorbed withabout 20% of the applied dose being absorbed percutaneously even after repeatapplications of the soap solution.

The results of a percutaneous absorption study with diapers washed in a solution containingtriclosan suggest that the absorption of triclosan through the skin was very low in rabbits[Ciba-Geigy, 1977b (26)]. It should be noted that in this study triclosan was not applieddirectly to the skin and that the amount of triclosan in the skin was not determined. Inanother percutaneous absorption study in rabbits, the extent of absorption of triclosan wasdependent on the type of formulation (solutions: >85% of the applied dose was absorbed;creams: 29 to 48% was absorbed; soap solutions: 2 to 10% was absorbed), as was

observed in studies with rats [Ciba-Geigy, 1976b (25)].

In a small percutaneous absorption study in dogs (n=3), triclosan in water (200 mg/kg) wasapplied to the shaven nuchal skin of dogs once daily for 2 weeks [Hong et al., 1976 (24)].Triclosan blood levels were 130 and 165 ppb (ng/mL) on Day 7 and 14, respectively. Thetotal extent of absorption could not be determined, as levels in the urine, faeces, tissues,and expired air, were not reported. Another study in the dog investigated the absorption oftriclosan through the oral mucosa [Lin et al ., 1994 (28)]. In this study, peak blood levels,which occurred within 6 to 8 hours, represented 0.7 to 2.7% (mean = 1.4%) of the applieddose. Daily urinary excretion was in the range of 1 to 4% (mean = 2%) of the applieddose. There were no apparent sex differences in plasma concentrations or urinary excretionof triclosan in dogs. Again, the total extent of absorption was not determined.

The percutaneous absorption of unlabelled triclosan was investigated in a pilot study and a90-day study with infant Rhesus monkeys [Parkes, 1978a (29); Hazleton Laboratories,1979b (30)]. In the pilot study, triclosan was detected in all blood samples following asingle dermal exposure to a soap solution containing triclosan (1 mg/mL, 0.1%), with bloodlevels detected up to 24 hours, and peak levels observed at 8 to 12 hours. In the 90-daystudy, only the glucuronide and sulfate conjugates were detected in blood samples, theglucuronide predominating in the early blood samples (Days 1 to 2), and triclosan sulfatepredominating in all subsequent blood samples (samples were taken daily for the 90-dayduration of the study). Triclosan was excreted in the urine primarily as the glucuronideconjugate, but was excreted in the faeces primarily in the free or unconjugated form. Lowlevels of triclosan were detected in tissues. The results of this monkey study indicate thattriclosan was absorbed percutaneously following 90 days of daily washing with 15 mL ofsoap (1 mg triclosan/mL) and that the proportion of plasma glucuronide and sulphateconjugates altered following chronic administration.

Summary of Dermal/Percutaneous Absorption Data

In summary, data from the percutaneous absorption studies conducted with triclosanindicate that it was relatively well absorbed through the skin in all species tested. Theextent of absorption was dependent on the formulation in which it was delivered (e.g.,greater absorption was observed following application of triclosan in solution than in acream or Vaseline formulation) and the duration of time that the applied dose remained onthe skin (e.g., lather/rinse off vs. apply/leave on). In the rat, the extent of percutaneous

absorption was approximately 23 to 28% of the applied dose of triclosan in ethanol,



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ethanol/ water, soap suspension, or a cream formulation (although higher absorption wasobserved in some studies).

3.3.5. Repeated dose toxicity

A number of repeated dose toxicity studies have been conducted for triclosan in mice, rats,hamsters, rabbits, dogs, and primates.

3.3.5.1. Repeated dose (14 days) dermal toxicity studies

Three short-term dermal toxicity studies were conducted in mice and rats for the purposesof establishing dose ranges for larger studies of longer durations. The major findings fromthese GLP dose range finding studies are presented in Table 10. Triclosan was tested in 2mouse studies using different vehicles (acetone and propylene glycol). Using a similar studydesign, rats were administered triclosan in an acetone vehicle.In the 2 mouse studies, triclosan was administered at dose levels of 0, 0.3, 0.6, 1.5, 3.0, or6.0 mg/animal/day for 14 days in acetone [Ref.: 8] or in propylene glycol [Ref.: 9]. Thesedermally-applied doses correspond to systemic doses of approximately 12, 24, 60, 120, or240 mg/kg body weight/day for a 25 g mouse (triclosan concentrations of 0.3, 0.6, 1.5, 3,and 6 % in 0.1 mL application volume). The triclosan was applied to a 2 x 3 cm2 hairlessarea on the dorsal side of the animal. Appropriate untreated and vehicle controls were used.As these were dose range finding studies, although GLP-compliant, they were not entirelycompliant with OECD guidelines for dermal studies (clinical chemistry, haematology, orurinalysis investigations were not conducted, and macroscopic and microscopic evaluationswere limited). Both mouse studies showed similar dose-related dermal and liver findingssuch as dermal irritation, increased liver weight, coagulative necrosis and centrilobularhypertrophy. Dose-related dermal findings started at 1.5 mg / animal / day (60 mg/kgbw/d) for triclosan in propylene glycol and at 3.0 mg / animal / day for triclosan in acetonevehicle. Liver effects were observed in animals treated with dermal doses of ≥ 1.5 mg /animal / day (≥ 60 mg/kg bw/d). The NOAEL is 24 mg/kg bw/d.

Ref.: 8, 9

In rats, a similar dose range finding study was conducted using an acetone vehicle.Dermally-applied doses of 0.3, 0.6, 1.5, 3.0, or 6.0 mg/day in the rat study correspond tosystemic doses of approximately 1.2, 2.4, 6, 12, or 24 mg/kg body weight/day for a 250 grat (triclosan concentrations of 0.1, 0.2, 0.5, 1, and 2 % in 0.3 mL application volume).Skin irritation such as erythema was observed at 6.0 mg/day, with findings in one femaleanimal at 1.5 mg/day considered to be incidental. Other related signs of skin irritation suchas eschar, and oedema were also noted. Histopathology of the erythema, scaling, andeschar showed acanthosis and hyperkeratosis at the highest dose. Gross pathology revealeddark areas of the liver in a few treated animals (not dose-related); however, no change inorgan weight was observed and no histopathology was associated with the gross pathology

findings.Ref.: 10

In summary, these 3 rodent dermal studies revealed a similar pattern of toxicity withrespect to dose-related dermal irritation and hyperkeratosis at the site of application. Inaddition, liver-related effects such as increased organ weight associated with centrilobularhypertrophy were observed in both mouse studies but not in the rat, indicating a speciesdifference in response to triclosan. The NOAEL was determined to be 0.6 mg/day (24 mg/kgbody weight/day) in both mouse studies, based on liver effects observed at doses of ≥ 1.5mg / animal / day. The NOAEL in the rat study was determined to be 3.0 mg / animal / day(12 mg/kg bw/day), based on dermal irritation at the highest dose of 6.0 mg / animal / day.



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Table 10: Findings from GLP Short-Term Repeated Dose Dermal Toxicity Studies forTriclosan

Species

(Strain)

Dosing Regimen

and Duration

Major Findings Reference;

GLP and

OECD Status

Mouse(CD-1)

0, 0.3, 0.6, 1.5,3.0, or 6.0mg/animal/d inacetone for 14days, in a volumeof 100 µL, appliedto 2x3 cm2 area onthe dorsal areaonce daily(triclosanconcentrations of0, 0.3, 0.6, 1.5, 3,and 6%)

Dermal irritation such as erythema was observed at the siteof application. Oedema, fissuring, eschar was observed atthe 2 highest doses which correlated with hyperkeratosis.Acanthosis was also observed in males at 1.5 mg/d. The livershowed dose-related increase in absolute and relative liverweight in females at 1.5 and in both sexes at 3.0 and 6.0mg/d. This increase associated with centrilobularhypertrophy. Mononuclear infiltrate was also observed butonly at the high dose. The NOAEL is considered to be 0.6mg/d. This corresponds to 24 mg/kg bw per day

Burns, 1997a(8)

GLP:compliant

OECD:comparable

Mouse(CD-1) 0, 0.3, 0.6, 1.5,3.0, or 6.0mg/animal/d inpropylene glycol for14 days, in avolume of 100 µL,applied to 2x3 cm2 area on the dorsalarea once daily(triclosanconcentrations of0, 0.3, 0.6, 1.5, 3,and 6%)

Dose-related dermal irritation was observed at the site ofapplication starting at 1.5 mg/d. Dose-related increases inabsolute and relative liver weights in both sexes at 6.0 mg/dand in males at 0.3, 1.5 and 3.0 mg/d. Centrilobularhypertrophy at doses ≥1.5 mg/d. The statistical increase inmale liver weights at 0.3 mg/d was not correlated with anyhistopathology changes. The NOAEL is considered to be0.6 mg/d. This corresponds to 24 mg/kg bw per day

Burns, 1996(9);

GLP:compliant

OECD:comparable

Rat(Crl:CDBR)

0, 0.3, 0.6, 1.5,3.0, or 6.0

mg/animal/d inacetone for 14days, in a volumeof 300 µL, appliedto 2x3 cm2 on thedorsal area oncedaily (triclosanconcentrations of0.1, 0.2, 0.5, 1,and 2%)

Dose-related skin irritation such as erythema was observedat 6.0 mg/d, with findings in 1 animal at 1.5 mg/d

considered incidental. Other related signs of skin irritationsuch as eschar, oedema were also noted. Histopathology ofthe erythema, scaling and eschar showed acanthosis andhyperkeratosis at the highest dose. Gross pathologyrevealed dark areas of the liver noted in a few treatedanimals (not dose-related); however, no change in organweight was observed and no histopathology accompaniedthis gross finding. The NOAEL was estimated byinvestigators to be 3.0 mg/animal/d equivalent to 12 mg/kgbw per day.

Burns, 1997b(10);

GLP:compliant

OECD:comparable

3.3.5.2. Repeated dose (21 days) inhalation toxicity

The inhalation toxicity of triclosan after 14 days of repeated dose administration wasevaluated in the rat. This study was performed prior to GLP regulations and theestablishment of OECD guidelines

In this study, groups of 10 male and 10 female rats were initially exposed to triclosan atconcentrations ranging from 50 to 1,300 mg/m3. Following the first day, due to theoccurrence of deaths, dyspnoea and general clinical signs indicative of poor health in thetreated animals, the test concentrations were reduced to 0, 50, 115, or 301 mg/m3 fordosing on Days 2-15, i.e., the remainder of the study. Although there were 12 unscheduleddeaths related to high-dose level exposure, 11 of the 12 rats died during Day 1 as a resultof the very high initial doses. Necropsy revealed congestion, inflammatory changes inmucous membranes and nasal cavity.In the remaining animals, dose-related increases in leukocyte count and alterations inserum chemistry such as glutamic-pyruvic transaminase, alkaline phosphatase were



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observed. Slight focal inflammation of the mucous membranes was observed in high-doseanimals at the end of the study. The NOAEL for inhaled triclosan was determined in thisstudy to be 50 mg/m3, the lowest dose tested.

Ref.: 32

3.3.5.3. Repeated dose (28 days) oral toxicity study in mice

A GLP-compliant 28-day repeated dose oral toxicity study of triclosan was conducted in themouse, using OECD Guideline 407 for the design of the study. In this mouse study, thesignificant findings were in the liver, which showed reversible hepatocellular hypertrophyand focal necrosis in mice that received the high dose (136 and 169 mg/kg body weight/dayin males and females, respectively).

Ref.: 31

A brief summary of the major findings from the repeated dose oral toxicity study ispresented in the table below.

Table 11: Findings from Short-Term Repeated Dose Oral Toxicity Studies for Triclosan

Species

(Strain)

Dosing Regimen

and Duration


GLP andOECD

Status

Mouse(MAGf)

0, 50, or 1,000 ppmin the diet (0, 6.5, or136 mg/kg bw/d inmales and 0, 8.3, or169 mg/kg bw/d infemales) for 28 dayswith 14 daysrecovery

Slight, but significant reversible decreases in erythroidparameters. Biochemistry showed significant increases (2-to 3-fold) in liver function enzymes in plasma (high-doseanimals); changes were almost completely reversed by theend of 14-d recovery. Slight changes in urea (high-doseanimals) and creatinine (high-dose females) were not fullyreversed. No macroscopic findings, but histopathologicalexamination showed liver changes in high-dose animals,including hepatocyte hypertrophy, hepatocyte necrosis

(focal) bordered by macrophages in some cases. SomeKupffer cells in the area contained a pigment that wasassumed to be iron. These changes were reversed in 14-dayrecovery period. Low-dose animals showed no histologicalchanges or changes in haematology or blood chemistry(except for a slight, not significant increase in liver functionenzymes in males of the low-dose group). Electronmicroscopy of selected livers of high-dose animals showedreversible proliferation of smooth endoplasmic reticulum andincrease in number and/or size of peroxisomes. No NOAELwas determined by investigators; however, it should benoted that no adverse effects were observed at the low dose(50 ppm).

Ciba-Geigy,1987(31);

GLP:compliant

OECD:No.407

consistent

CommentA reversible decrease in phosphate was observed in females of both doses and liverenzymes were slightly increased but not significant in low dose males.

3.3.5.4. Sub-chronic (90 days) oral toxicity studies

The safety of triclosan has been evaluated in sub-chronic oral toxicity studies in mice, rats,hamsters, rabbits, dogs, and baboons, using either dietary administration or capsules. Testspecies were evaluated for clinical observations, body weight, body weight gain, food andwater consumption, haematological, clinical chemistry, ophthalmological and urinalysisparameters, macroscopic observations, and microscopic findings. In addition, theseinvestigations included a wide range of dose levels for triclosan. Pivotal studies conducted in

mice, rats and hamsters were conducted pursuant to GLP and generally followed OECDguidelines. Findings from these studies are presented in Table 12 (GLP- and OECD-compliant studies). Rabbit, dog, and baboon studies were conducted prior to the



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establishment of GLP standards and OECD guidelines. Findings from these studies arepresented in Table 13 (non-GLP and/or non-OECD compliant studies).In the pivotal studies conducted in compliance with GLP and following OECD guidelines for90-day (sub-chronic) oral toxicology studies, the highest doses tested were 900 mg/kgbody weight/day in the mouse, 600 mg/kg bw/day in the rat, and 900 mg/kg bw/day in thehamster.

Subchronic oral toxicity study in mice

In the GLP mouse study, CD-1 mice (15/sex/group) were administered triclosan via the dietat dose levels corresponding to 0, 25, 75, 200, 350, 750, or 900 mg/kg bw/day for 13weeks. Additional satellite groups (10/sex/group), for interim sacrifice at 7 weeks, wereadministered 0, 25, 350, and 900 mg/kg body weight/day. This study contained a GLP andOECD compliance statement and was well designed and conducted. The results of this studyindicated that the liver was the primary target organ in mice. Treatment-related increases inalkaline phosphatase were observed in females and males, at ≥25 mg/kg bw/day and at≥200 mg/kg body weight/day, respectively. Decreased erythrocyte count and haemoglobinwere noted at ≥25 mg/kg bw/d (male) and all animals at ≥75 mg/kg bw/d. Other changes

noted included decreased total cholesterol in all animals at 25 mg/kg bw/day and higherand increased alanine aminotransferase (males at 350 mg/kg body weight/day and above,females at 750 mg/kg body weight/day and above). Increased gamma glutamyltransferasewas observed in both males and females at 750 mg/kg body weight/day. Dose-relatedincreases in absolute and relative liver weights were observed in both males and femalesstarting at 75 mg/kg bw/day. Gross and histopathology findings in the liver includedenlarged dark or thickened lobes, with histomorphologic centrilobular hepatocellularhypertrophy, vacuolization, pigment accumulations, necrosis, and/or inflammation. Thesehepatic changes were noted in males at 75 mg/kg bw/day and in both males and females atall higher doses. In the more severe cases of hepatocellular hypertrophy, hepatocytes wereindividualized, although the overall hepatic architecture was still intact. In addition to liverfindings, increased extramedullary haematopoiesis was observed in the spleen of animals at

750 and 900 mg/kg bw/day and, at doses of 200 mg/kg body weight/day and higher,hyperplasia in male glandular stomachs and inflammation in female kidneys occurred. Nohistomorphologic alterations were observed in males at 25 mg/kg bw/day or in females at25 or 75 mg/kg bw/day. A NOAEL was not established from this study since treatment-related changes in haematology parameters, increased alkaline phosphatase, and decreasedcholesterol were observed at the low dose.

Ref.: 33

Subchronic oral toxicity study in rats

In a 90-day GLP toxicity study in rats that served as a dose range-finding study for a 2-yearoral carcinogenicity study, triclosan (FAT 80’023) was administered to Sprague-Dawley rats(25/sex/group) via the diet at concentrations of 0, 1000, 3000, or 6000 ppm(approximately 0, 100, 300, or 600 mg/kg bw/day based on calculations of mean bodyweight, food consumption and target dose level. No test article-related mortality occurred.Decreased body weight along with gradually decreased food consumption was observed atthe high-dose groups. Diet spillage at study initiation occurred at all treatment groups.Treatment-related effects on erythrocyte parameters RBC, haemoglobin and haematocritwere observed in mid- and high-dose groups. Increased ketones were found in high-dosemales and decreased triglycerides in high-dose males and females. Interim necropsyfindings, conducted at 45 days, revealed increased liver weight changes in males (mid- andhigh-dose) and females (high-dose only). At terminal necropsy, absolute liver weight forhigh-dose males and relative liver weight for both male and female dose groups wereincreased. Kidneys weights were increased in males at the highest dose and spleen weightswere decreased in mid-and high-dose males. Histopathologic examination revealed mild

hepatic centrilobular cytomegaly and fatty methomorphosis in mid- and high-dose males.These changes were also common to female rats, but occurred at a lower frequency. No



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histomorphologic changes were observed in the spleen. The low dose was not associatedwith any treatment-related findings; thus, the NOAEL was determined to be 1,000 ppm(~100 mg/kg body weight/day).

Ref.: 34

Subchronic oral toxicity study in hamsters

In the 13-week GLP hamster study, triclosan (FAT 80’023) was administered to SyrianGolden Hamsters (15/sex/treatment group; 20/sex/control, 10/sex/group for interimsacrifice at 7 weeks) via the diet at doses of 0, 75, 200, 350, 750, or 900 mg/kg bodyweight/day. Treatment was not associated with any mortality or clinical signs. Decreasedbody weight gain was observed at 750 and 900 mg/kg body weight/day. Food consumptionwas decreased in males at 900 mg/kg body weight/day and females at 350 mg/kg bodyweight/day and above. Water consumption was increased at all groups given 200 mg/kgbody weight/day and higher. Increased coagulation times, changes in red cell morphologyand red cell indices indicated microcytic type anaemia in high-dose animals (750 and 900mg/kg body weight/day). Clinical chemistry disturbances of liver and kidney function were

observed at doses of 750 mg/kg body weight/day and 900 mg/kg body weight/day.Biologically significant clinical chemistry changes noted in alkaline phosphatase and alanineaminotransferase indicated possible liver toxicity; however, organ weight determinationsand macroscopic and microscopic examination revealed no corresponding findings. The maintarget organ toxicity in hamsters was dose-related nephrotoxicity (tubular casts, tubularbasophilia, tubular dilation). Although the LOEL may be estimated to be 200 mg/kg bodyweight/day, study investigators determined the NOEL to be 75 mg/kg body weight/day dueto increased water consumption and some changes in urinalysis parameters.

Ref.: 35

Comment of the SCCPThe NOAEL is set at 200 mg/kg bw/d based on nephrotoxicity indicated by microscopic

findings and polyuria, haemoglobinuria and haematouria.



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Table 12: Findings from GLP Sub-Chronic Oral Toxicity Studies for Triclosan

Species(Strain)

DosingRegimen and

Duration

Major Findings Reference;GLP and

OECD

StatusMouse(CD-1)

0, 25, 75, 200,350, 750, 900mg/kg bw/d for13 weeks via dietaryadmixture.Interim sacrificeat 7 weeks.

Conducted pursuant to OECD Guideline No. 408. Clinicalsigns at highest dose. Decreased food consumption anddecreased body weight at ≥750 mg/kg bw/d.Increased alkaline phosphatase in females at ≥25 mg/kgbw/d and males at ≥200 mg/kg bw/d. Decreased totalcholesterol at ≥25 mg/kg bw/d, increased alanineaminotransferase (males at ≥350 mg/kg bw/d, females at≥750 mg/kg bw/d). GGT increased≥750 mg/kg; however,not statistically significant. Decreased erythrocyte countand haemoglobin at ≥25 mg/kg bw/d (male) and allanimals at ≥75 mg/kg bw/d. Increased liver weight andliver to body weight ratio at doses of ≥75 mg/kg bw/d inthe main study and at doses of ≥350 mg/kg bw/d atinterim sacrifice. Decreased kidney weights were observed

in males at ≥350 mg/kg bw/d and females at 900 mg/kgbw/d. Enlarged dark/thickened lobes correlated withhistomorphologic centrilobular hepatocellular hypertrophy,vacuolization, pigment accumulations, necrosis and/orinflammation noted in males at 75 mg/kg bw/d and allanimals at 200, 350, 750 mg/kg bw/d. Extramedullaryhaematopoiesis was observed in the spleen of higher dose(≥750 mg/kg) animals. No histomorphologic alterationswere observed in males at 25 mg/kg bw/d and in femalesat 75 mg/kg bw/d. A NOAEL could not be determined.

Trutter,1993(33);

GLP:compliant

OECD:No.408consistent

Rat(Sprague-Dawley)

0, 1,000, 3,000,or 6,000 ppm via diet (~ 0, 100,300, or 600mg/kg bw/d) for

90 days via dietaryadmixture.Interim sacrificeat Day 45.

Conducted pursuant to OECD Guideline No. 408. Highdoses were accompanied by decreased body weight withgradual decreased food consumption and diet spillage atinitiation for all treatments. Treatment-related effects onerythrocyte parameters were observed. Interim necropsy

revealed increased liver weight changes in males (mid- andhigh-dose) and females (high dose only). At terminalnecropsy, liver weight for high-dose males and relativeliver weight for both male and female dose groups wereincreased. Histopathologic examination revealed mildhepatic centrilobular cytomegaly and fatty methomorphosisin high and mid-dose males. These changes were alsocommon to female rats; however, at a lower frequency.The NOAEL was considered to be 1,000 ppm (~100 mg/kgbw/d).

LittonBionetics,1983(34);

GLP:compliant




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Species(Strain)

DosingRegimen and

Duration


OECD

Status

Hamster(SyrianGolden)

0, 75, 200, 350,750 or 900mg/kg bw/d for13 weeks via dietaryadmixture.Interim sacrificeat Week 7.

Conducted pursuant to OECD Guideline No. 408.Decreased body weight gains were observed at ≥750mg/kg bw/d. Food consumption was decreased in males at900 mg/kg bw/d and females at ≥350 mg/kg bw/d. Waterconsumption was increased at all groups given 200 mg/kgbw/d and higher.Increased coagulation times, changes in red cellmorphology and red cell indices in high-dose animals(≥750 mg/kg bw/d). Slightly increased alanineaminotransferase levels in females at the highest dose, andin both sexes increased cholesterol at the highest dose. At≥750 mg/kg bw/d, decreased levels of alkalinephosphatase were observed. At 900 mg/kg bw/d,decreased gamma glutamyltransferase was observed.Urinalysis revealed polyuria, haemoglobinuria andhaematouria in all animals given ≥350 mg/kg bw/d.Interim and terminal necropsy revealed no change in anymajor organ weight in females. In males, increased

absolute and relative kidney weights at 750 mg/kg bw/dafter 13 weeks and 900 mg/kg bw/d at the interim andterminal necropsy. Associated with these findings was tandiscolour and/or granulated kidneys. Histopathologyshowed nephrotoxicity (e.g., tubular casts, tubularbasophilia, tubular dilation) which was dose-related withrespect to incidence and severity; microscopic changeswere noted starting at the dose of 350 mg/kg bw/d.Stomach inflammation (gastritis, glandular erosions) wasalso observed at ≥750 mg/kg bw/d. Based on changesnoted in water consumption, which were not consideredadverse, the LOEL was determined to be 200 mg/kg bw/d;study investigators determined the NOEL to be 75 mg/kgbw/d.At 750 and 900 mg/kg/day, the kidneys were identified as

a target organ based on macroscopic, histopathologic andclinical findings. Clinical chemistry changes also indicatedliver effects; however, no microscopic findings wereobserved. The red blood cell also showed treatment-related effects. At 350 mg/kg/day, microscopic findings ofnephrotoxicity were observed. The NOEL was determinedby study investigators to be 75 mg/kg bw/day.

Schmid etal ., 1994(35);

GLP:compliant


Subchronic oral toxicity study in rabbits

The results of 2 studies in rabbits were inconsistent; in 1 study, triclosan was reported to bewell-tolerated, with no treatment-related effects up to the dose level of 125 mg/kg bw/day,

whereas in a second study, the NOAEL was determined to be 3 mg/kg bw/day, as animalsgiven doses of 30 or 150 mg/kg bw/day showed pulmonary infections. However, it shouldbe noted that study investigators stated that the relationship of the lung findings totriclosan was unclear.

Ref.: 36, 37

Subchronic oral toxicity study in dogs, study 1

Inconsistency also was found in a comparison of 2 sub-chronic dog studies, the first ofwhich showed haematology, clinical chemistry, macroscopic and microscopic findings at alldoses tested, including some effects at the lowest dose tested of 25 mg/kg bw/day. As a

result, no NOAEL was determined for the study. Ref.: 38



SCCP/1192/08


Subchronic oral toxicity study in dogs, study 2

In a subsequent study, where doses were lowered to 0, 5, 12.5, or 25 mg/kg bodyweight/day, there were no treatment-related findings at any dose, even though the highestdose tested in the second study was the same as the lowest dose tested in the first study.

Ref.: 39

Subacute & subchronic oral toxicity study in baboons

In baboon studies 2 – 20 animals per dose level were administered 0, 1, 3, 10, 30 and 100mg/kg via oral gelatine capsules for 4 or 13 weeks (6 animals 3 mg/kg bw/d), respectively.Symptoms such as agitation, anger and aggression were observed in the one femalereceiving 100 mg/kg. No other clinical signs were observed in other treatment groups. Notreatment-related changes were found in ophthalmoscopic examinations and in waterconsumption, haematology and body weight. Findings were similar among terminalhistopathological examination of treated groups from 4 weeks and 13 weeks of treatment.Evidence of chronic interstitial pneumonitis was seen throughout all animals. Lymphocyticinfiltration was seen in the liver and large intestines. These findings were not different

among control and treated animals. Ref.: 40

Long term (1 year) oral toxicity study in baboons

A total of 56 animals (3/sex/dose (main study) plus 2/sex/dose (interim, 6 months) plus2/sex/dose (recovery group, 4 weeks after dosing) were used. Animals were dosed orally,once daily, with prefilled capsules with 0, 30, 100, or 300 mg/kg bw/d triclosan. Thecontrols received 1 capsule containing 600 mg lactose + 0.5% magnesium stearate per day.Vomiting was observed in some mid and high dose animals accompanied by an increasedincidence of low food intake. Deterioration of condition and abdominal pain was observed inHigh Dose animals. Incidence of diarrhoea was increased in Mid Dose animals and greatly

increased in High Dose animals. No significant effects in Males were observed except at 39and 52 weeks, when decreased WBC noted in Mid Dose and High Dose groups. Femalesshowed slight decreases in erythroid parameters at all time points for High Dose group, butonly significant up to 26 weeks. Also decreased WBC was observed in High Dose Females at52 weeks (not significant). Decrease in absolute brain weight and increases in kidney andliver weights (relative to BW) in High Dose animals were significant when Male and Femaledata combined (data at termination). NOEL is estimated to be 30 mg/kg bw/d based on theabsence of any effect, including diarrhoea, in baboons at the low dose level in this study.

Ref.: 41

CommentIn baboon studies conducted at doses up to 300 mg/kg bw/d, both 4/13-week and 1-yearinvestigations showed time-dependent haematology findings. Incidental clinical chemistrychanges were observed; however, there was no evidence of hepatic or renal injuryaccompanying these findings [Ref. 40; 41]. Clinical signs observed in the longer-term studywere not observed in the 4/13-week study. Significant differences in study design as wellas quality of investigation likely contributed to discrepancies between these studies.



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Table 13: Findings from Non-GLP, Non-OECD Sub-Chronic Oral Toxicity Studies forTriclosan

Species

(Strain)

Dosing

Regimen and

Duration


GLP and

OECDStatus

Rabbit (NewZealandWhite)

0, 12.5, 25, 62.5or 125 mg/kgbw/d for 90 daysvia dietaryadmixture.

Conducted prior to OECD guidelines, but considered similar indesign. Terminal examination revealed no organ weightchanges or gross pathology findings. Histopathologyexamination found no differences in high-dose animalscompared to control animals. Incidental microscopic changesnoted in high-dose animals included granulomatousinfiltrations in lungs in 1 female and 2 males and nephrosis in1 high-dose female. Overall, triclosan was well tolerated at alldoses. No NOAEL was determined in this study, in which thehighest dose used did not produce treatment-related effects.

Leuschneret al .,1970a(36);

PredatesGLP andOECD

OECD:comparable

Rabbit

(Albino)

0, 3, 30, 150

mg/kg bw/d for13 weeks via oralgavage.

Conducted prior to OECD guidelines, but considered similar in

design. Dose-related mortality was observed at 30 and 150mg/kg bw/d. At doses of 30 and 150 mg/kg bw/d,neutrophilia and lymphopenia were observed on variousobservation days but were not consistent throughout thestudy.The lung (which was associated with macroscopic andmicroscopic changes) appeared to be the target organ.Pulmonary infection was observed in 3/6 rabbits at 30 mg/kgbw/d and 3/6 rabbits from 150 mg/kg bw/d. Perirenal abscessin 1 rabbit was observed at 30 mg/kg bw/d. Limited organweight determinations showed no treatment-related findings.Gross macroscopic findings in the lung corresponded withhistopathologic lung lesions, oedema in 30 and 150 mg/kgbw/d treated animals and lung necrosis in 2 high-doseanimals. No such histomorphology changes were noted incontrol or 3 mg/kg bw/d animals. Based on findings ofneutrophilia and lymphopenia at 30 mg/kg bw/d and absenceof histomorphologic alterations, 3 mg/kg bw/d was determinedto be the NOAEL. However, the study authors stated that therelationship of the lung lesions, infection, oedema andsometimes necrosis observed at the 2 highest doses to testarticle administration is unclear. Dosing accidents orregurgitation with resultant pulmonary infection wassuggested.

Paterson,

1969(37);

PredatesGLP andOECD

OECD:comparable

Dog (Beagle) 0, 25, 50, 100 or200 mg/kg bw/dvia gelatinecapsules for 91days.

Conducted prior to OECD guidelines, but considered similar indesign, with the exception that clinical pathology evaluationswere limited. Seven unscheduled deaths (1-25 mg/kg bw/d,2-100 mg/kg bw/d and 4-200 mg/kg bw/d). Severity ofdiarrhoea was dose-related. Haematology evaluationsrevealed decreased haemoglobin, PCV and red blood cells and

increased ESR and reticulocytes in dogs at ≥50 mg/kg bw/d aswell as during interim evaluation in moribund dogs. Increasedalkaline phosphatase in all animals at doses of ≥50 mg/kgbw/d. Additionally, high SGOT and SGPT were noted in theseanimals. Bile salts, polymorphonuclear leukocytes wereobserved in dogs at ≥25 mg/kg bw/d. Terminal organ weightdeterminations revealed increased liver, pancreas and adrenalweights for 100 and 200 mg/kg bw/d animals.Bile retention, necrosis, pathological fat and unusual Kupffercell activation were observed histopathologically in the liver.In animals which showed severe liver damage, the bonemarrow was hyperplastic. In 2 dogs given 200 mg/kg bw/dand one dog at 100 mg/kg bw/d, focal interstitial nephritis wasobserved. Convoluted epithelium of the kidney was observedin 2 dogs given 25 mg/kg bw/d but not observed at any otherdose.Based on the findings in this study, no NOAEL was determined.

Paterson,1967(38);

PredatesGLP and

OECD

OECD:comparable



SCCP/1192/08


Species(Strain)

DosingRegimen and

Duration


OECD

Status

Dog (Beagle) 0, 5, 12.5 or 25mg/kg bw/d via diet for 90 days.

Conducted prior to OECD guidelines, but considered similar indesign. Occasional pasty to thin faeces were noted in allgroups. Haematology, clinical chemistry and urinalysisevaluations were limited to high-dose group animals.Macroscopic evaluations found no gross pathology for anyorgan. Organ weight measurements showed no changes inrelative or absolute weights. Histopathologic evaluation of allmajor organs from all animals showed no difference amongtreated and control animals. No NOAEL was determined in thisstudy, in which the highest dose used did not producetreatment-related effects.

Leuschneret al .,1970b(39);

PredatesGLP andOECD

OECD:comparable

Baboons(papiocynocephalusand anubis)

4-week dosing:0, 1, 10, 30, or100 mg/kg bw/dvia gelatinecapsules.

13-week dosing:0 or 3 mg/kgbw/d via gelatinecapsules.

Conducted prior to OECD guidelines, but consideredcomparable in design (but limited in number of parametersevaluated). Blood chemistry revealed no haematologychanges. Urinalysis showed no changes. Clinical chemistrychanges included high plasma urea levels in 2 animals at 30mg/kg bw/d, 2 animals at 3 mg/kg bw/d, 1 control baboon

and increased SGPT in individual male baboons at 1 mg/kgbw/d and 3 mg/kg bw/d. Terminal necropsy after 4 weeks andafter 13 weeks showed no organ weight differences amongtreated and controls. No gross macroscopic findings wereobserved. After 4 weeks of treatment, minor changes such asdark nodules in the large intestine wall at ≥10 mg/kg bw/d.Adhesion in lung surface and rib cage, slight thickening ofcapsule of the liver in 10 mg/kg bw/d female and 1 mg/kgbw/d male, and control male and female were observed.Limited conclusions can be derived from this study due toinsufficient number of animals evaluated. A NOAEL was notdetermined for this study.

Noel et al .,1969(40);

PredatesGLP and

OECD

OECD:comparable

Baboons(papio)

0, 30, 100, or300 mg/kg bw/dvia capsules daily

for 1 year.Necropsies werescheduled at 6months, 12months, and 13months (28 dayspost-treatment).

Conducted prior to OECD guidelines, but considered similar indesign (but limited in number of parameters evaluated).Vomiting and diarrhoea occurred at mid and high-doses.

Haematology parameters showed decreased white blood cellsin males at Week 29 and 52 in mid and high-dose groups. Infemales, slight decreases in erythroid parameters at all timepoints were observed for the high-dose group (statisticallysignificant only up to Week 26). Incidental changes inpotassium and sodium were observed. Slight changes, bothincreases and decreases in SGOT and AP were observed;however, not considered treatment-related. No differenceswere found in urinalysis.At necropsy, decreased absolute brain and increases in kidneyand liver weights were observed in high-dose animals.Histopathologic examination found similar histology in bothtreated and control animals.The NOEL was determined to be 30 mg/kg bw/d.

Ciba-Geigy,1975a(41);

PredatesGLP andOECD

OECD:comparable

3.3.5.5. Sub-Chronic Dermal Toxicity

The sub-chronic dermal toxicity of triclosan has been investigated in rats, dogs, andmonkeys. The studies conducted in weanling dogs and newborn Rhesus monkeys were notGLP-compliant and not conducted pursuant to OECD guidelines. The pertinent details fromthese studies are summarized in Tables 14 (GLP rat study) and 15 (non-GLP, non-OECDstudies).

Sub-Chronic Dermal Toxicity (90 d) in rats

The rat study was conducted according to both GLP and OECD guidelines (Crl:CDBR

(VAF/Plus) strain). IRGASAN DP300 (0, 10, 40, 80 mg/kg bw/d) diluted in propylene glycolwas applied to clipped, unabraded dorsal surface of each animal (approx. 10% of total body



SCCP/1192/08


surface) at dose volume of 2.0 mL/kg daily (approximately 0.5 to 4.0% triclosan). Theappropriate dose was applied under a gauze pad and covered. Daily contact exposure wasfor at least 6 hours. Dermal observations of erythema and/or showed oedema in all treatedgroups. No treatment-related changes were found in ophthalmoscopic examinations and inwater consumption and body weight. Occult blood was observed in the urine of high-doseand satellite male rats and to a lesser extent in mid-dose males and females. Isolated

changes were observed in erythrocyte parameters in high dose animals. Small butstatistically significant changes were observed in some serum chemistry parameters. NOAEL= 80 mg/kg/day (excluding dermal irritation)

Ref.: 11

Sub-Chronic Dermal Toxicity (90 d) in dogs

Weanling dogs exposed to triclosan through dermal application for 90 days of doses rangingfrom 2 to 200 mg/kg bw/day in a non-GLP study showed no toxicity except for dermalirritation at the highest dose tested.

Ref.: 12

Sub-Chronic Dermal Toxicity (90 d) in monkeys

The major findings from a 90-day bathing study conducted in newborn Rhesus monkeysshowed that repeated exposure to triclosan (0.1% in a soap solution, 5 min exposure) waswell-tolerated. No treatment-related toxicities were observed.

Ref.: 13

Table 14: Findings from GLP Sub-Chronic Dermal Toxicity Studies for Triclosan

Species

(Strain)

Dosing Regimen

and Duration


GLP and

OECDStatus

Rat (Crl:CDBRVAF/Plusstrain)

0, 10, 40, or 80mg/kg bw/d inpropylene glycolapplied undergauze for at least6 hours (90days). Triclosanwas applied toapprox. 10% oftotal body surfacein a volume of2 mL/kg bw.Approximatetriclosan testconcentrations

were ~0, 0.5, 2,and 4% for a0.25 kg animal.A recovery groupwas included atthe high dose.

Conducted pursuant to OECD Guideline No. 411. Dermalerythema and/or oedema was observed in all treatment groups.Haematology parameters showed isolated changes in erythrocyteparameters in high-dose animals; however, these findings werewithin expected range. Similarly, clinical chemistry findingsshowed small but statistically significant changes in serumchemistry. Occult blood in urine was observed in the high doseand satellite male rats and to a lesser extent in mid-dose femalesand males. Histopathology examinations observed eschar anddesquamation, hyperplasia/hyperkeratosis of epidermis, dermalinflammation and focal necrosis observed at all doses. Reversal ofthe dermal effects was seen during the 28-day recovery period.Microscopic changes in the urinary bladder of 3 males wereobserved. Coagulative necrosis of hepatocytes was also observed.Both findings lacked a dose-response. With respect to general

toxicity, the NOAEL was determined to be 80 mg/kg bw/d (thehighest dose tested). No NOAEL for dermal toxicity wasdetermined.

Trimmer,1994(11);

GLP:compliant

OECD:compliant



SCCP/1192/08


Table 15: Findings from Non-GLP, Non-OECD Sub-Chronic Dermal Toxicity Studies forTriclosan

Species

(Strain)

Dosing Regimen

and Duration


GLP and

OECDStatus

Dog(Beagleweanling)

0, 2, 20, or 200mg/kg bw/dapplied to ashaved area ofthe neck for 90days (12.5%triclosan added to50% corn-starchvehicle w/vol;size of applicationarea notreported.)

Conducted prior to OECD guidelines and missing a number ofstudy parameters. Upper respiratory disease during the study upto Day 25. Diarrhoea in all animals was not attributed to the testsubstance. Dermal irritation (dermatitis) was observed at the highdose and was dose-dependent. There were no treatment-relatedbiochemical or haematological changes. Microscopic examinationrevealed no histomorphology changes in the skin. Test substancewas reported to show “little toxic effect” aside from dermalirritation (pathology data not reported).

Dorner,1973(12);

PredatesGLP andOECD

Monkey(Rhesus,newborn)

0.1% soapsolution, latheredfor 5 minutes andthen washed for90 days (size ofapplication areanot reported).Recovery group:30 days withoutbathing.

Conducted prior to OECD guidelines and not comparable to currentOECD guidelines (e.g., exposure method). Mild anaemia, redblood cell and haemoglobin values showed variations betweenanimals, but was not considered treatment-related. Thesechanges were attributed to frequent sampling. Limited clinicalchemistry parameters were evaluated due to limitations in bloodsampling; however, of the parameters measured, no consistentchanges were noted. Termination examination showed no organweight changes. Histopathologic evaluation revealed similarfindings of focal adrenal mineralisation, pulmonary infiltration, andlow-grade pneumonia in both treated and control animals.Hepatocellular vacuolar changes and hepatic extramedullaryhaematopoiesis were observed in both treatment and controlanimals. No histological changes were observed in skin sectionstaken for examination.No differences were noted between animals sacrificed after 90days of treatment and recovery animals. This 0.1% triclosan soapsolution was well-tolerated under the conditions of this study.

HazletonLabs,1979a(13);

PredatesGLP andOECD

OECD: noapplicableguidelines

3.3.5.6. Chronic (> 1 year) Toxicity

Chronic toxicity was assessed in the carcinogenicity studies with rats, mice and hamsters(See also 3.3.7).

Long Term Toxicity/Carcinogenicity study – 18 Months (Mouse)

Animals were dosed daily via the diet for 544-552 days in total. Dietary admixtures weremixed weekly for 13 weeks, then every 4 weeks; amounts of triclosan were adjusted usingmost recent weekly body weight and feed consumption data. 0, 10, 30, 100, or 200 mg/kgbw/d in the diet (control, low dose, low mid dose, high mid dose, or high dose).A significant decrease in survival was observed in High Mid Dose males and high dosefemales whereas high dose males the decrease was slight but not significant.No adverse effects on Body weight, food consumption and urine parameters attributable tothe test substance were observed. In biochemistry significant changes at 18 months wereincreased (230-560%) liver function enzymes in high Mid Dose and High Dose Males andFemales; decreased (75-90%) cholesterol in all treated Males and Females; decreasedbilirubin (up to 67%) in Females (all doses except Low Dose). In haematology: small(<15%) but significant dose-related changes in erythroid parameters (Males and Females).Increased % reticulocytes (males) and platelets (males and females; 25-37%, dose-related)

significant at 18 months. Increased WBC, neutrophils, & lymphocytes at higher doses in



SCCP/1192/08


Males (6 months) were not considered indicative of test substance toxicity by the studyauthors. No changes were recorded at low Dose.Dose-related, significant increases in liver weights were observed in males and females atlow Mid Dose, high Mid dose and High Dose. Increase in incidence of hepaticnodules/masses and/or discolorations in Males and Females of high Mid Dose and High Dosegroups compared to control. Slight increase in testicular germinal epithelium

degeneration/atrophy was observed at High Dose (only controls and High Dose examined).No treatment-related histopathological changes other than in liver. Hepatocyte hypertrophywas present in all animals except Low Dose Females. Brown pigment in hepatocytes inhigher dose rats was found to be lipofuscin and iron. The LOAEL was 10 mg/kg bw/d basedon liver changes. This dose level was considered as NOAEL based on haematotoxicity whenexcepting the target organ liver.

Ref.: 66

Long Term Toxicity/Carcinogenicity study – 104-week (Rat)

Animals were dosed daily via the diet at concentrations of 0, 300 (Low Dose), 1,000 (MidDose), 3,000 (High Dose) ppm in 104-week study. An extra group of rats was given a

“toxic” dose of 6,000 ppm and killed at 52 weeks. Doses were calculated weekly based onfood intake and weekly mean BW. At 52 weeks, calculated doses were 0, 12, 40, and 127mg/kg bw/d (Males) and 0, 17, 56, and 190 mg/kg bw/d (Females), respectively. The doseof 6,000 ppm gave doses of 247 (Males) or 422 (Females) mg/kg bw/d.No treatment-related effects on mortality were observed. Body weight was significantlydecreased in High Dose Females up to week 52. Food Consumption was generallysignificantly increased in High Dose Males. In biochemistry transient changes in protein,BUN, glucose, bilirubin, triglyceride and liver enzymes were found.In haematology slight (4-10%) but significant changes in erythroid parameters (red bloodcell count in Males and Females), including in Low Dose Males rats at week 104 wererecorded. Also increases in mean corpuscular haemoglobin concentration was observed infemales at mid and high dose and in males in addition at low dose. Slightly increased

altered red cell morphology was observed in High Dose Males. Increased clotting time (HighDose Males), decreased WBC (33%) in Females was measured at 104 weeks. Decreases in% monocytes were transient (High Dose Females) but significant at 104 weeks (High DoseMales).At 52 weeks slight relative weight decreases at High Dose were observed: liver, brain,kidneys (Males) and heart, ovaries and spleen (Females). At 104 weeks increased absoluteadrenal weight (Mid Dose Males) and decreased brain weight (High Dose Males); increasedabsolute and relative ovary weight (High Dose Females), decreased relative spleen weight(High Dose Females), and decreased absolute and relative spleen weights (Mid DoseFemales).Morphology revealed hepatocyte hypertrophy and hepatocytic inclusions (hyaline-staining,round-shaped) in Male rats: significant – High Dose (13 weeks), 6,000 ppm (52 weeks). Noother histological lesions were considered to be treatment-related. The study authorsconsidered the NOAEL as ~48 mg/kg/d for both sexes, combined (~56 mg/kg bw/d inFemales and ~40 mg/kg bw/d in Males).

Ref.: 67

CommentSCCP considers the NOAEL as 12 mg/kg bw/d due to haematoxicity and decreased absoluteand relative spleen weights (Mid Dose Females).

Long Term Toxicity/Carcinogenicity study – 95 Weeks (Hamster)

The animals were administered 0, 12, 75, or 250 mg/kg bw/d FAT 80’023/S. in the diet for90 weeks. Survival was significantly decreased in males and was generally poor (38-58%)

in females at 90 weeks. Body weight gain was significantly decreased in all high-dosehamsters and was accompanied by a slight (3%), but significant decrease in food



SCCP/1192/08


consumption in high-dose females. Biochemical changes observed at termination includedincreases (<50%) in blood urea nitrogen in high-dose hamsters and in triglycerides in mid-and high-dose males. Haematological changes observed included slight (<15%), butsignificant decreases (but not dose-related) in erythroid parameters in mid- and high-doseanimals, increased white blood cells in high-dose animals, and increased lymphocytes inhigh-dose females at termination. At termination, renal nephropathy was observed in

animals of all dose groups, with increased incidence and severity at the high-dose level. Inaddition, males showed atypical hyperplasia in the stomachs (fundic region), along withspermatozoa and germ cell effects at the high dose. One high-dose female showed atypicalhyperplasia in the fundic region that was considered to be treatment-related. High-dosefemales showed distended gastric glands and a few treated females of all doses showedbenign papillomas of the non-glandular region of the stomach (not discussed in report).Hepatic effects were few, with only rarified hepatocytes reported in a few male animals(6/60 in high dose vs. 3/121 in controls).The NOAEL was set as 75 mg/kg bw/d.

Ref.: 68

Comment on setting a NOAEL

In the Table below the derived NOAELs from subchronic and chronic studies in differentspecies were compiled.

EPA in its recent evaluation selected the NOAEL of the baboon study (30 mg/kg bw/d) forrisk assessment based on clinical signs of toxicity which are presumably due to oraltreatment. This might not be relevant for cosmetic uses.

The applicant in its safety evaluation used the NOAEL of the 95-week study in hamsters asthis species was judged to be the most relevant to humans based on pharmacokinetics(75 mg/kg bw/d). Alternatively as a more conservative value, the NOEL of the 104-week ratstudy (≈ 48 mg/kg bw/d for both sexes) was used.

SCCP considers the NOAEL of this long term toxicity study in rats as 12 - 17 mg/kg bw/d (≈ 14.5 mg/kg bw/d) due to haematoxicity and decreased absolute and relative spleenweights. Haematoxicity was also detected in the 13-week subchronic oral toxicity studies inmice and rats, in hamsters only at higher doses and in the 1-year toxicity study in baboons.This was further confirmed by changes in haematology parameters in the long term studiesin mice and hamsters. Interestingly, also in the 13-week subchronic dermal toxicity study inrats changes in erythrocytes parameters were observed.The SCCP will use the NOAEL of 12 mg/kg bw/d of the long term toxicity study in rats forrisk assessment.

Subchronic oral toxicity

study in mice A NOAEL was not established from this study since treatment-related changes inhaematology parameters, increased alkaline phosphatase, and decreased

cholesterol were observed at the low dose 25 mg/kg body weight/day.Subchronic oral toxicity

study in rats The low dose was not associated with any treatment-related findings; thus, theNOAEL was determined to be 1,000 ppm (~100 mg/kg body weight/day).

Subchronic oral toxicity

study in hamsters

The NOAEL is set at 200 mg/kg bw/d based on nephrotoxicity indicated bymicroscopic findings and polyuria, haemoglobinuria and haematouria.

Long term (1 year) oraltoxicity study in baboons

NOEL is estimated to be 30 mg/kg bw/d based on the absence of any effect,including diarrhoea, in baboons at the low dose level in this study.

Long Term Toxicity /Carcinogenicity study – 18

Months (Mouse)

The LOAEL was 10 mg/kg bw/d based on liver changes. This dose level wasconsidered as NOAEL based on haematotoxicity when excepting the target organliver.

Long Term Toxicity /Carcinogenicity study –

104-week (Rat)

The study authors considered the NOAEL as ≈ 48 mg/kg/d for both sexes,combined (≈ 56 mg/kg bw/d in Females and ≈ 40 mg/kg bw/d in Males).SCCP considers the NOAEL as 12 mg/kg bw/d due to haematoxicity anddecreased absolute and relative spleen weights (Mid Dose Females).

Long Term Toxicity /

Carcinogenicity study – 95Weeks (Hamster)

The NOAEL was set as 75 mg/kg bw/d.



SCCP/1192/08


3.3.6. Mutagenicity / Genotoxicity

3.3.6.1. Mutagenicity / Genotoxicity in vitro

Bacterial gene mutation assay

Study 1

Guidelines: /Species/Strain: Salmonella typhimurium TA92, TA98, TA100, TA1535, TA1537Replicates: triplicates in a single testTest substance: FAT 80 023/ASolvent: DMSOBatch: /Purity: /Concentrations: 0.1, 0.3, 0.9, 2.7 and 8.1 µg/ml without metabolic activation

Additionally 24.3 and 72.9 µg/ml without metabolic activation for TA920.1, 0.3, 0.9, 2.7, 8.1, 24.3 and 72.9 µg/ml with metabolic activation

Treatment: Plate incorporation method with 48 h incubation timeGLP: /Date: May 1978

The Ames-test was performed with the bacterial tester strains Salmonella typhimurium TA92, TA98, TA100, TA1535 and TA1537 with and without S9-mix. Liver S9 fraction fromAroclor 1254-induced rats was used as exogenous metabolic activation system. Toxicity wasevaluated on the basis of a reduction in the number of spontaneous revertant colonies. Theexperiment was performed with the direct plate incorporation method. Justified negativeand positive controls were concurrently tested.

ResultsThe test compound did not induce an increase in the number of revertant colonies in anystrain at any concentration tested both in the presence or absence of metabolic activation.

ConclusionUnder the experimental conditions used FAT 80 023/A was not mutagenic in the genemutation tests in bacteria both in the absence and the presence of S9 metabolic activation.

Ref.: 42

CommentThe test is performed before the implementations of OECD guidelines. Batch number andpurity were not reported. The test has only limited value.

Study 2

Guideline: OECD 471Strain: Salmonella typhimurium TA98, TA100, TA1535 and TA1537Replicates: 3 replicates in 2 individual experiments both in the presence and

absence of S9-mix.Test substance: Triclosan [Irgasan DP 300; 2,4,4’-trichloro-2’-hydroxy diphenyl]Solvent: DMSOBatch: S 15155 TO1Purity: > 99%

Concentrations: 0.015, 0.05, 0.15, 0.5 and 1.5 μg/plate both without and with S9-mixMetabolic activation: Experiment 1: 3% and 10%Experiment 2: 10% and 30%



SCCP/1192/08


Treatment: pre-incubation method with 60 minutes incubation and a selectionperiod of 72 h.

GLP: In complianceDate: March – September 1988

Triclosan was investigated for the induction of gene mutations in Salmonella typhimurium

(Ames test). Test concentrations were based on the results of a preliminary toxicity studywith concentrations up to the (nowadays) prescribed maximum concentration of 5000μg/plate. Toxicity was evaluated on the basis of a substantial reduction in the number ofmicro-colonies in the background bacterial lawn. The experiments were performed with thepre-incubation method. Liver S9 fraction from Aroclor 1254-induced rats was used asexogenous metabolic activation system. Negative and positive controls were in accordancewith the OECD guideline.

ResultsIn the preliminary toxicity study triclosan was toxic towards the tester strains at the higherdoses and consequently 1.5 μg/plate was chosen as the top dose level. In the mainexperiments toxicity was reported at the top dose level. No substantial increases in

revertant colony numbers in any of the tester strains were observed at any of the dosestested either in the absence or presence of metabolic activation.

ConclusionUnder the experimental conditions used triclosan was not genotoxic (mutagenic) in the genemutation tests in bacteria both in the absence and the presence of metabolic activation.

Ref.: 43

Study 3

Guideline: OECD 471

Strain: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA1538Replicates: triplicates both in the presence and absence of S9-mixTA100: triplicates in 2 retests in the absence of S9-mix

Test substance: 39316Solvent: DMSOBatch: CC# 14663-09Purity: /Concentrations: Experiment 1: 0.00167, 0.005, 0.0167, 0.05, 0.1 and 0.167 μg/plate

without S9-mix0.05, 0.167, 0.5, 1.67, 2.5 and 5 μg/plate with S9-mix

Experiment 2 and 3: 0.000167, 0.0005, 0.00167, 0.005, 0.01, 0.0167,0.0333, 0.05, 0.1 and 0.167 μg/plate without S9-mixfor TA100 only.

Treatment: direct plate incorporation with 48 incubation without and with S9-mixGLP: In complianceDate: March 1993

Test compound 39316 was investigated for the induction of gene mutations in Salmonellatyphimurium (Ames test). Test concentrations were based on the results of a preliminarytoxicity pre-screen with concentrations up to the prescribed maximum concentration of5000 μg/plate evaluating the growth of the background lawn and/or frequency ofspontaneous revertants. The experiments were performed with the direct plateincorporation method. Liver S9 fraction from Aroclor 1254-induced rats was used asexogenous metabolic activation system. Negative and positive controls were in accordance

with the OECD guideline.



SCCP/1192/08


ResultsToxicity was reported at the higher doses tested. However, due to excessive toxicity forstrain TA100 without S9-mix only three acceptable dose levels remained. In a retest astatistical significant but not dose dependent increase in revertant colonies was observed.This finding was not confirmed in a second retest. Biologically relevant, dose dependentincreases in the number of revertants were also not observed in the other strains at any of

the doses tested both in the absence or presence of metabolic activation.

ConclusionUnder the experimental conditions used test compound 39316 was not genotoxic(mutagenic) in the gene mutation tests in bacteria both in the absence and the presence ofS9 metabolic activation.

Ref.: 44

CommentThe test was not repeated. Purity of test substance 39316 (triclosan) was not reported.

Mutagenicity test with S a cc h a r om y c e s c e r e v i s i a e

Study 1

Guideline: /Strain: Saccharomyces cerevisiae MP-1Replicates: triplicatesTest substance: FAT 80 023/ASolvent: DMSOBatch: /Purity: /Concentrations: 10, 20, 30, 40, 50, 60 and 200 mg/l

Treatment: 3.5 h treatment, followed by an expression period of 4-6 days for inter- and intragenic recombinants or 8 days for cycloheximide resistance.

GLP: /Date: November 1978

FAT 80 023/A was investigated for the induction of gene mutations in a mutagenicity testwith Saccharomyces cerevisiae. Test concentrations were based on the results of apreliminary toxicity study. 4-nitroquinoline-N-oxide served as positive control. For thedetermination of inter- and intragenic recombinants yeast cells were cultured on normal andtryptophan free agar, for the determination of cycloheximide resistance, an indication offorward mutations, on cycloheximide agar.

ResultsIn the main experiment no colonies were found at the highest dose as a result of theinhibitory effect of FAT 80 023/A on the growth of the yeast cells. Treatment with FAT 80023/A did not result in a significant increase in the incidence of intergenic recombinants norin an increase in cycloheximide resistance. The occasional increases in intragenicrecombinants in the mid doses of 30 and 40 mg/l were not dose dependent and can beattributed to variation inherent in the test system

ConclusionUnder the experimental conditions used FAT 80 023/A was not mutagenic in thismutagenicity test in yeast.

Ref.: 45

Comment



SCCP/1192/08


The test is performed before the implementations of OECD guidelines. Batch number andpurity of FAT 80 023/A (triclosan) were not reported. The test has only limited value.

Study 2

Guideline: /Strain: Saccharomyces cerevisiae MP-1Replicates: 20 replicates in 3 experimentsTest substance: Irgasan DP 300 [5-chloro-2-(2,4-dichlorophenoxy)-phenol]Solvent: DMSOBatch: /Purity: 99.7% isomer pureConcentration: 0.2 mg/mlTreatment: 3.5 h treatment, followed by an expression period of 4 days for inter-

and intragenic recombinants or 8 days for actidione resistance.GLP: /Date: June 1978

Irgasan DP 300 was investigated for the induction of gene mutations in a mutagenicity testwith Saccharomyces cerevisiae. Only one test concentration was used. For thedetermination of inter- and intragenic recombinants yeast cells were cultured on normal andtryptophan free agar, for the determination of actidione resistance, an indication of forwardmutations, on actidione agar. A positive control was not included.

ResultsThe results demonstrated that Irgasan DP 300 had an effect in the mutation and intergenicrecombination system but not on the intragenic recombination system.

Conclusion

Under the experimental conditions used Irgasan DP 300 was mutagenic in this mutagenicitytest in yeast.

Ref.: 46

CommentThe test is performed before the implementations of OECD guidelines. Batch number wasnot reported. Only one dose was tested. The test has only very limited value.

I n v i t r o gene mutation assay with Mouse Lymphoma cells

Study 1

Guideline: /Species/strain: Mouse lymphoma cell line L5178Y/tk +/- Replicates: Duplicates, two independent testsTest substance: FAT 80 023/ASolvent: 0.05 N NaOHBatch: /Purity: /Concentrations: 15.8 µg/ml (18h treatment) and 28.9 µg/ml (4 h treatment) without

metabolic activationTreatment: 4 or 18 h treatment and an expression period of 3 days. The selection

period was not mentioned (probably 12 days)

GLP: /Date: May 1978



SCCP/1192/08


FAT 80 023/A was assayed for gene mutations at the tk locus of mouse lymphoma cellsboth in the absence and presence of S9 metabolic activation. Test concentrations werebased on the results of a toxicity test on cell survival. The concentration required to produce80% cell-kill was calculated. In the main test, cells were treated for 4 or 18 followed by anexpression period of 3 days to fix the DNA damage into mutations. The incidence of mutantswas determined with methothrexate, cytosine arabinoside and thymidine as antimetabolites.

Negative and positive controls were not included.

ResultsFollowing treatment with FAT 80 023/A an increase in the mutant frequency was notobserved at both dose levels tested in the absence of S9-mix.

ConclusionUnder the experimental conditions used FAT 80 023/A was not mutagenic in mouselymphoma cells in vitro.

Ref.: 49

Comment

The test is performed before the implementations of OECD guidelines. Toxicity was notmeasured in the main experiment. Batch number and purity of FAT 80 023/A (triclosan)were not reported. The test has only limited value.

Study 2

Guideline: /Cells: L5178Y mouse lymphoma cellsReplicates: duplicate cultures in 2 independent experimentsTest substance: TriclosanSolvent: DMSO

Batch: S15155 TO1Purity: > 99%Concentrations: Experiment 1: 5.0, 7.5, 10.0, 15.0, and 20.0 µg/ml without S9-mix

3.5, 7.5, 10.0 and 15.0 µg/ml with S9-mixExperiment 2: 2.5, 5.0, 7.5, 10.0 and 15.0 µg/ml without S9-mix

1.0, 5.0, 7.5, 10.0 and 15.0 µg/ml with S9-mixTreatment 3 h treatment without and with S9-mix; expression period 48 h and

selection period of 12 daysGLP: In complianceDate: September 1988

Triclosan was assayed for gene mutations at the tk locus of mouse lymphoma cells both inthe absence and presence of S9 metabolic activation. Test concentrations were based onthe results of a preliminary toxicity test considering suspension growth. In the main test,cells were treated for 3 h in the absence or presence of S9-mix followed by an expressionperiod of 48 h to fix the DNA damage into a stable tk mutation. Liver S9-mix fraction fromAroclor 1254-induced rats was used as exogenous metabolic activation system. Toxicity wasmeasured in the main experiments as mean % survival relative to the solvent controlcultures. Negative and positive controls were included.

ResultsIn experiment 2 without S9-mix and in experiment 1 with S9-mix the highest dose testedshowed the appropriate level of toxicity (10-20% adjusted relative total growth after thehighest dose). In the experiment 1 without S9-mix and in experiment 2 with S9-mixexcessive toxicity was seen at the highest dose but the first analysable doses did show the

appropriate level of toxicity.



SCCP/1192/08


A more or less dose dependent increase in the mutant frequency was found in allexperiments. However, the increase in mutant frequency was either less than 2-fold thebackground mutant frequency or for concentrations with a mutant frequency higher thanthe 2-fold back ground level the relative survival exceeded the 10%.

Conclusion

Under the experimental conditions used, triclosan was considered not mutagenic in themouse lymphoma assay at the tk locus.

Ref.: 50

CommentsThe test was not performed according the OECD guideline.

I n v i t r o unscheduled DNA synthesis test

Study 1

Guideline: /Species/strain: primary rat hepatocytesReplicates: triplicate subculturesTest substance: TriclosanSolvent: DMSOBatch: S15155 TO1Purity: /Concentrations: Experiment 1: 0.6, 1.3, 2.5, 5, 10, 20, 40 and 80 μg/ml

Experiment 2: 0.16, 0.3, 0.6, 1.3, 2.5, 5, 10 and 20 μg/mlTreatment: 18 - 20 h treatmentGLP: In complianceDate: May - September 1988

Triclosan was investigated for the induction of unscheduled DNA synthesis (UDS) in primaryhepatocytes of rats. Hepatocytes were isolated from male Fischer F344 rats. Hepatocyteswere exposed simultaneously to triclosan and 10 μCi/ml 63H-thymidine for 18 - 20 h. Aftertreatment cultures were divided in 3 subcultures for quantification of UDS whereas a fourthsubculture was used to determine the toxicity of each treatment. Evaluation ofautoradiography was done 7 days after exposure.UDS was measured by counting nuclear grains and subtracting the average number ofgrains in 3 nuclear-sized areas adjacent to each nucleus; this value is referred to as netnuclear grain count. Unscheduled synthesis was determined in 50 randomly selectedhepatocytes. Negative and positive controls were included.

ResultsIn experiment 1 at concentrations above 10 μg/ml no viable cells were seen at the end ofthe exposure period. In experiment 2 at 10 μg/ml only 14% survival was observed at theend of exposure whereas at 20 μg/ml no viable cells were seen at all. Althoughautoradiography was performed on all treated cultures, the autoradiographs of 20, 40 and80 μg/ml of experiment 1 and of 20 μg/ml of experiment 2 were not scored due toexcessive toxicity.At none of the remaining dose levels in both experiments neither an increased net nucleargrain count, nor a notable increase in cells with 6 or more nuclear grains per cells, nor anotable increase in cells with 20 or more nuclear grains per cells (cells in repair) wasobserved.

Conclusion

Under the experimental conditions used triclosan did not induce unscheduled DNA synthesisin primary rat hepatocytes and, consequently, is not genotoxic in the in vitro UDS test.



SCCP/1192/08


Ref.: 52

CommentThe test was not performed according the OECD guideline. Purity was not reported. The testhas only limited value.

Study 2

Guideline: /Species/strain: primary rat hepatocytesReplicates: triplicate culturesTest substance: 39317Solvent: DMSOBatch: /Purity: /Concentrations: 0.25, 0.5, 1 and 2.5 μg/mlTreatment: 18 - 20 h treatment

GLP: In complianceDate: March - April 1993

39317 was investigated for the induction of unscheduled DNA synthesis (UDS) in primaryhepatocytes of rats. Hepatocytes were isolated from male Fischer F344 rats. Hepatocytes,grown on cover slips, were exposed simultaneously to 39317 and 10 μCi/ml 3H-thymidine(specific activity 50 - 80 Ci/mM) for 18 - 20 h. Test concentrations were based on theresults of a preliminary screen on precipitation of 39317. Evaluation of autoradiography wasdone 7 days after exposure. Coverslips from each dose were pre-screened for toxicity byvisual inspection under a microscope.UDS evidenced as a net increase of grains over the nucleus was quantified by determiningthe nuclear and cytoplasmic grain counts. Cytoplasmic grain count was performed in 3

nuclear-sized areas adjacent to each nucleus. Unscheduled synthesis was determined in 150nuclei per dose point. Negative and positive controls were included.

ResultsAnalytical results of the test article performed by the sponsor indicated that the actualconcentrations were 0.3, 0.6, 1.18 and 3 μg/ml.At none of the dose levels an increased net nuclear grain count nor an increase in cells with5 or more nuclear grains per cells (cells in repair) was observed.

ConclusionUnder the experimental conditions used 39317 did not induce unscheduled DNA synthesis inprimary rat hepatocytes and, consequently, is not genotoxic in the in vitro UDS test.

Ref.: 53

CommentThe test was not performed according the OECD guideline. The test was not repeated. Batchnumber and purity were not reported. However, stability and purity were reported as “theresponsibility of the sponsor”.

I n v i t r o chromosome aberration test

Study 1

Guideline: OECD 473 (1983)

Species/strain: Chinese hamster ovary (CHO-K1 –BH4) cells

Replicates: duplicates in a single test



SCCP/1192/08


Test substance: TriclosanSolvent: DMSOBatch: S15155 TO1Purity: > 99%Concentrations: 0.1, 0.3, 0.5 and 1.0 µg/ml without metabolic activation

4.8, 9.5, 19 and 30 µg/ml with metabolic activation

Treatment: 24 h treatment without S9-mix.6 h treatment with harvest time 24 h after start of treatment with S9-mix

GLP: in complianceDate: April – August 1988

Triclosan has been investigated in the absence and presence of metabolic activation for theinduction of chromosomal aberrations in CHO cells. Test concentrations were chosen on thebasis of the results of a preliminary toxicity test measuring cell death and decline in mitoticindex. In the absence of S9 cells were treated for 24 h and immediately harvested; in thepresence of S9 cells were treated for 6 h and harvested 24 h after the start of treatment.Two hours before harvest, each culture was treated with colchicine solution (final

concentration 0.25 μg/ml) to block cells at metaphase of mitosis. Liver S9 fraction fromAroclor 1254-induced rats was used as exogenous metabolic activation system.Chromosome (metaphase) preparations were stained with 10% Giemsa and examinedmicroscopically for chromosomal aberrations. Negative and positive controls were inaccordance with the OECD guideline.

ResultsIn both the absence and the presence of a metabolic activation system, triclosan did notcause a statistical increase in cells with chromosome aberrations.

ConclusionUnder the experimental conditions used triclosan did not show evidence for a genotoxic

(clastogenic) activity in CHO cells in vitro. Ref: 47

CommentIt is not known whether in the main test the cells were sufficiently exposed since the mitoticindex was not determined. Yet, in the experiment with metabolic activation a dosedependent increase was seen with a 4-fold increase in the highest dose compared to theuntreated control. Therefore the SCCP consider this test as equivocal. The test was notrepeated. The results of this test can only be used as supportive evidence.

Study 2

Guideline: OECD 473, 1983Species/strain: Chinese hamster V79 cells

Duplicates in a single experimentTest substance: FAT 80’023/QSolvent: ethanolBatch: EN 91390.76Purity: /Concentrations: 0.1, 1.0 and 3.0 µg/ml without and with S9-mixTreatment: 4 hours with harvest times 7 (high dose only), 18 and 28 h (high dose

only) after the start of treatment.GLP: in complianceDate: February – December 1990



SCCP/1192/08


FAT 80’023/Q was investigated in the absence and presence of metabolic activation for theinduction of chromosomal aberrations in V79 cells. Test concentrations were chosen on thebasis of the results of a pre-experiment for toxicity test measuring reduction in platingefficiency. Cells were treated for 4 h. Harvest times were 7 h (high dose only), 18 h or 28 h(high dose only) after the beginning of treatment. Liver S9 fraction from Aroclor 1254-induced rats was used as exogenous metabolic activation system. Two hours (7 h harvest

time) or 2.5 h (18 and 28 h harvest times) before harvest, each culture was treated withcolchicine solution (final concentration 0.2 μg/ml) to block cells at metaphase of mitosis.Chromosome (metaphase) preparations were stained with Giemsa and examinedmicroscopically for chromosomal aberrations. Negative and positive controls were inaccordance with the OECD guideline.

ResultsIn the cytogenetic experiment, the mitotic index was reduced after treatment with thehighest concentration at each fixation interval, except at interval 18 and 28 h in thepresence of S9-mix. At the harvest time 7 h (both without and with S9) and 28 h (with S9)no increase in cells with chromosomal aberrations was observed.A dose dependent and biologically relevant increase in cells with chromosomal aberrations

was found at the harvest times of 18 h (both without and with S9-mix) and 28 h (withoutS9-mix).

ConclusionUnder the experimental conditions used FAT 80’023/Q induced an increase in the number ofaberrant cells and, consequently, is mutagenic (clastogenic) in V79 cells in vitro.

Ref: 48

CommentExposure of the cells (measured as a decrease in the mitotic index) was not determined inthe main test. In the protocol, it is mentioned that at harvest time 28 h only the high dose(3.0 µg/ml) is tested. However, in the table with the result at harvest time 28 h without S9-

mix, (table 22 of the study report) the test article concentration is reported as 1.0 µg/ml.The test was not repeated. Purity of FAT 80 023/A (triclosan) was reported as “cf. AnalyticalCertificate in sponsor’s file”.

Summary: genotoxicity/mutagenicity of triclosan in vitro

strain/cell type test

compound concentration S9 result quality ref

Bacterial gene mutation assay

TA92, TA98, TA100,TA1535, TA1537

FAT 80 023/A 0.1-8.1-(72.9) µg/plate

0.1-72.9 µg/plate

-

+

negative

negative

limitedvalue

42

TA98, TA100, TA1535,TA1537

triclosan 0.015-1.5 µg/plate - /+

negative appropriate 43

TA98, TA100, TA1535,TA1537, TA1538

39316 0.000167-0.167µg/plate

0.05-5 µg/plate

-

+

negative

negative

sufficient 44

Yeast gene mutation assay

S. cerevisiae MP-1 FAT 80 023/A 10-200 mg/l - negative limitedvalue

45

S. cerevisiae MP-1 Irgasan DP 300 0.2 mg/ml - mutagenic limitedvalue

46

Gene mutation assay in mammalian cells

L5178Y/tk +/-

mouselymphoma cells FAT 80 023/A 15.8-28.9 µg/ml - negative limitedvalue 49



SCCP/1192/08


strain/cell type test

compound concentration S9 result quality ref

L5178Y/tk +/- mouselymphoma cells

triclosan 2.5-20 µg/ml

1-15 µg/ml

-

+

negative

negative

appropriate 50

Unscheduled DNA Synthesis test

primary rat hepatocytes triclosan 0.16-80 µg/ml - negative limitedvalue 52

primary rat hepatocytes 38317 0.25-2.5 µg/ml - negative sufficient 53

Chromosome aberration test

CHO cells triclosan 0.1-1 µg/ml

4.8-30 µg/ml

-

+

negative

negative

supportiveevidence

47

V79 cells FAT 80’023/Q 0.1-3 µg/ml - /+

mutagenic appropriate 48

3.3.6.2. Mutagenicity / Genotoxicity in vivo

Host mediated assay with S. t y p h i m u r i u m in mice

Guideline: /Species: albino NMRI miceBacteria: Salmonella typhimurium TA98, TA100, TA1535 and TA1537Group sizes: 6 mice/groupTest substance: FAT 80 023/ASolvent: 2% CMCBatch: /Purity: /Dose levels: 50, 100, 200 and 400 mg/kg bw

Route: oral gavageCell removal: 1 h after the injection of the bacteriaGLP: /Date: March 1979

FAT 80 023/A was investigated for the induction of gene mutations in the host mediatedassay with Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537. After beingfasted for 16 h the mice were treated orally by gavage 2 h, 1 h and immediately beforebacteria were injected. The bacteria were injected in the lateral vein of the tail. One h afterthe injection of the bacteria the mice were killed, the livers removed and homogenised. Thehomogenate was centrifuged and the centrifugate was resuspended in saline. Five plateswith 0.2 ml of the undiluted samples were used for the determination of the mutant count.

The total bacterial count present in the animals was determined for each dosage or controlgroup as a whole. Dilutions of 10-5 and 10-6 of the pooled samples were spread on 5 and 4NB (nutrient brooth) plates respectively. The mutant rate is calculated from the mutantcount and the total bacterial count. The mutation factor is the ratio of the mean mutationrate for each dosage group to the mean mutation rate of the control group. Negative andpositive controls were not included.

ResultsAt the tested doses 50, 100, 200 and 400 mg/kg the mutation rates in comparison withthose of the control were not significantly increased in Salmonella typhimurium strainsTA98, TA100, TA1535 and TA1537

ConclusionUnder the experimental conditions used FAT 80 023/A was not mutagenic in this hostmediated assay with S. typhimurium in mice.



SCCP/1192/08


Ref.: 54

CommentThe test is performed before the implementations of OECD guidelines. Batch number andpurity of FAT 80 023/A (triclosan) were not reported. The test has only limited value.

Host mediated assay with mouse lymphoma cells in mice

Guideline: /Species: DBA/2f/Bom miceCell type: Mouse lymphoma cell line L5178Y/tk +/- Group sizes: 6 mice/groupTest substance: FAT 80 023/ASolvent: 0.05 N NaOHBatch no: /Purity: /Dose levels: 1313 mg/kg bw

Route: oral gavageCell removal: 6 days after cell inoculation and 3 days after compound administrationGLP: /Date: May 1978

FAT 80 023/A was investigated for the induction of gene mutations in the host mediatedassay. Test concentrations were based on the results of a toxicity test on cell survival. Themice were inoculated intraperitoneally with 106 cells per animal. FAT 80 023/A wasadministered orally. Six days after cell inoculation and 3 days after FAT 80 023/Aadministration, the cells were removed from the peritoneal fluid under a-septic precautions.The cells were seeded and cultured to fix the DNA damage into mutations. The incidence ofmutants was determined with methothrexate, cytosine arabinoside and thymidine as

antimetabolites. Negative and positive controls were not included.

ResultsAt 1313 mg/kg bw the target cell count was reduced with 50%. There was no increase inthe number of mutant colonies in comparison with the control.

ConclusionUnder the experimental conditions used FAT 80 023/A was not mutagenic the host mediatedassay with mouse lymphoma cells.

Ref.: 49

CommentThe test is performed before the implementations of OECD guidelines. Batch number andpurity of FAT 80 023/A (triclosan) were not reported. The test has only limited value.

Mouse spot test

Study 1

Guideline: /Species/strain: T stock males and C57BL/6JHan femalesGroup size: /Test substance: Irgasan DP 300Batch: /

Purity: /Dose level: 50 mg/kg bw



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Route: intraperitoneal injectionDay of administration: day 10 of pregnancyVehicle: Hanks balanced salt solutionGLP: /Date: August 1978

Irgasan DP 300 has been investigated for the induction of mutations in the mouse spot test.Female C57BL/6JHan mice were exposed by ip injection to a single dose of 50 mg/kg bwIrgasan DP 300 at day 10 of pregnancy. Between 2 and 5 weeks of age the F1 animals wereanalysed for the occurrence of spots.

ResultsThe examination was rendered difficult because with the exception of some midventralwhite spots all spots consisted of a more or less large mixture of mutant and non-mutanthairs.The frequency of colour spots in mice in the test and control groups clearly show thatIrgasan DP 300 in a dose of 50 mg/kg is active in the spot test. Compared to othercompounds the authors consider Irgasan DP 300 as a mutagen of only medium

effectiveness.

ConclusionUnder the experimental conditions used Irgasan DP 300 was mutagenic in the mouse spottest.

Ref.: 63

CommentThe test is performed before the implementations of OECD guidelines. Group size, batchnumber and purity were not reported. The test has only limited value.

Study 2

Guideline: /Species/strain: male T stock and female C57BL/E miceGroup size: /Test substance: Irgasan ®DP 300 (triclosan)Batch: /Purity: 99.7%Dose level: 0, 1, 2, 4, 8 and 25 mg/kg bwDay of administration: day 9.25 or 10.25 of pregnancyVehicle: 60% methanolScoring for mutations: day 12 after birthGLP: /Date: March - October 1979

Triclosan has been investigated for the induction of somatic mutations in the mouse spottest. Female C57B1/E mice were mated with T stock males. On days 9.25 or 10.25 postfertilisation triclosan was administered by ip injection. At 12 days after birth the offspringwas scored for coat colour spots. A positive control was not included.

ResultsTriclosan when injected in 60% methanol killed 12 out of 41 treated females of the 25mg/kg group. In the other treatment groups all animals survived. Prenatal survival wasclearly reduced by 25 mg/kg triclosan in both the 9.25 and 10.25-day treated groups. Levelof exposure below 25 mg/kg produced no obvious effects on prenatal survival. Postnatal

survival was severely reduced after 25 mg/kg and slightly but significantly reduced after 8



SCCP/1192/08


mg/kg in both the 9.25 and 10.25-day treated groups and significantly reduced after 4mg/kg (average exposure 3.2 mg/kg) only in the 10.25-day treated group.Within the 9.25 day group the incidence of recessive spots and midventral white spots is notincreased by triclosan treatment. Within the 10.25 day set triclosan has a clear effect on theincidence of midventral white spots at 25 mg/kg but no effect on the incidence of recessivespots in comparison to the control group. However, 25 mg/kg was markedly toxic both to

the mothers and the animals exposed in utero.

ConclusionUnder the conditions of this test, triclosan did not induce somatic mutations at non/subtoxic concentrations and, consequently, triclosan is not mutagenic in this mouse spot test.

Ref.: 64

CommentThe experiment is conducted before the development of the OECD guidelines. The batch nrwas not reported. The test can be used as supportive evidence.

Bone marrow chromosome aberration test in Chinese hamsters (C r i c e t u l u sg r i s e u s )

Study 1

Guideline: /Species: Female Chinese hamsters (Cricetulus griseus)Group sizes: 4 females/groupTest substance: GP 41 353 (triclosan)Batch: 3Purity: /Dose levels: 150, 300 and 600 mg/kg bw

Vehicle: 0.5 % CMCTreatment: oral by gavage, daily application on 2 consecutive days, 24 apart.Sacrifice times: 6 h after the last treatmentGLP: /Date: 1973

Triclosan has been investigated for the induction of chromosome aberrations in bonemarrow cells of Chinese hamsters. Chinese hamsters were exposed twice by oral gavage 24h apart on two consecutive days. The highest dose 600 mg/kg bw is approximately 1/3 ofthe LD50. Two h after the second treatment the animals were injected intraperitoneally with10 mg colcemid/kg. Bone marrow cells were collected 6 h after the last treatment. Bonemarrow preparations were stained with acetic-orcein and examined microscopically forchromosome aberrations. Cyclophosphamide was used as positive control.

ResultsIn comparison to the vehicle control there was no biologically relevant or statisticallysignificant enhancement in the number of cells with chromosome aberrations with any doselevel tested.

ConclusionUnder the experimental conditions used triclosan is not genotoxic (clastogenic) in bonemarrow cells of Chinese hamsters.

Ref.: 55

Comment



SCCP/1192/08


The test is performed before the implementations of OECD guidelines. Purity was notreported. There were no indications of toxicity and thus sufficient exposure is not proven.The test has only limited value.

Study 2

Guideline: /Species: Female Chinese hamsters (Cricetulus griseus)Group sizes: 6 male and 6 females/groupTest substance: FAT 80 023/ABatch: 652Purity: /Dose levels: 75, 150, 300 and 600 mg/kg bwVehicle: 0.7 % CMCTreatment: oral by gavage, thrice weekly for twelve weeks.Sacrifice times: 6 h after the last treatmentGLP: /

Date: February 1979

FAT 80 023/A has been investigated for the induction of chromosome aberrations in bonemarrow cells of Chinese hamsters after long term treatment. Chinese hamsters wereexposed thrice weekly by oral gavage for 12 weeks. The highest dose 600 mg/kg bw isapproximately 1/3 of the LD50. Two h after the last treatment the animals were injectedintraperitoneally with 10 mg colcemid/kg. Bone marrow cells were collected 6 h after thelast treatment. Bone marrow preparations were stained with acetic-orcein and examinedmicroscopically for chromosome aberrations. The solvent was used as negative control; apositive control was not included.

Results

Of the 12 animals treated with 600 mg/kg bw 5 died within the first week, additionally 3 bythe 3rd, 4th and 9th week. One of the animals in the low dose group died at the end of theexperiment.In comparison to the vehicle control animals, long term treatment with FAT 80 023/A didnot result in a biologically relevant or statistically significant enhancement in the number ofcells with chromosome aberrations with any dose level tested.

ConclusionUnder the experimental conditions used FAT 80 023/A is not genotoxic (clastogenic) in bonemarrow cells of Chinese hamsters.

Ref.: 56

CommentThe test is performed before the implementations of OECD guidelines. Long term treatmentis not a routinely performed protocol. Purity of test substance FAT 80 023/A (triclosan) wasnot reported. There were no data on toxicity and thus sufficient exposure is not proven. Thetest has only limited value.

Bone marrow chromosome aberration test in rats

Guideline: OECD 475 (1984)Species/strain: Wistar ratsGroup size: 5 rats/sex/groupTest substance: FAT 80’023/Q

Batch: EN 91390.76Purity: /



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Dose level: 4000 mg/kg bwRoute: oral, onceVehicle: 1% carboxymethylcellulose-suspensionSacrifice times: 6 h, 24 h and 48 h.GLP: In complianceDate: December 1990 – April 1991

FAT 80’023/Q has been investigated for the induction of chromosome aberrations in bonemarrow cells of rats. The test concentration was based on a pre-experiment for toxicitymeasuring acute toxicity. In the main experiment rats were exposed orally to 4000 mg/kgbw, the maximum tolerated dose. From approximately 18 h before treatment with the testcompound the animals were fasted. Bone marrow cells were collected 6 h, 24 h or 48 hafter dosing. Two and a half h before sacrifice animals were injected intraperitoneally withthe spindle inhibitor colcemid (2.0 mg/kg bw) to arrest cell in metaphase. To describe acytotoxic effect, and thus exposure of the target cells, the mitotic index (percentage cells inmitosis) was determined. Bone marrow preparations were stained with Giemsa andexamined microscopically for the mitotic index and chromosomal aberrations. Negative andpositive controls (24 h sacrifice time only) were in accordance with the OECD guideline.

ResultsFAT 80’023/Q did not induce a reduction in the mitotic index and is considered not cytotoxicfor bone marrow cells and thus exposure of the target cells is not proven. Exposure to 4000mg/kg bw FAT 80’023/Q did not induce a biological relevant increase in cells withchromosomal aberrations in bone marrow cells of the rat up to a sacrifice time of 48 h aftertreatment.

ConclusionUnder the experimental conditions used FAT 80’023/Q was not genotoxic (clastogenic) inbone marrow cells of rats.

Ref.: 57

CommentPurity of FAT 80’023/Q (triclosan) was reported as “see Analytical Certificate in sponsor’sfile”. Since the test substance did not induce a decrease in the mitotic index as compared tothe concurrent negative control data, evidence of exposure of the target cells is lacking andthus the test has only limited value.

Bone marrow micronucleus test in mice

Guideline: OECD 474 (1982)Species/strain: CD-1 miceGroup size: 5 mice/sex/groupTest substance: TriclosanBatch: S 15155 TO1Purity: ≥ 99%Dose level: 5000 mg/kg bwRoute: oral by gavage, onceVehicle: 1% methylcelluloseSacrifice times: 24 h, 48 h and 72 h after dosing.GLP: In complianceDate: March - August 1988

Triclosan has been investigated for the induction of micronuclei in bone marrow cells ofmice. The test concentration was based on a preliminary toxicity test with concentrations up

to the maximal prescribed dose of 5000 mg/kg bw recording cell death and signs ofmalreaction. In the main experiment rats were exposed orally to 5000 mg/kg bw. Following



SCCP/1192/08


dosing the animals were examined regularly and any mortalities or clinical signs of reactionto the test compounds were recorded. Bone marrow cells were collected 24 h, 48 h and 72h after dosing. Toxicity and thus exposure of the target cells was determined by measuringthe ratio between polychromatic and normochromatic erythrocytes (PCE/NCE ratio). Bonemarrow preparations were stained with 10% Giemsa and examined microscopically for thePCE/NCE ratio and micronuclei. Negative and positive controls were in accordance with the

OECD guideline.

ResultsIn the preliminary toxicity study mice treated with the highest dose (5000 mg/kg bw)showed slight piloerection, hunched posture, waddling and ptosis. One male mouse treatedwith the highest dose (5000 mg/kg bw) died. In the main test the mice only showed thefirst 6 h after exposure piloerection and hunched posture.At the sacrifice time 24 h and 48 h but not at 72 h triclosan induced a reduction in thePCE/NCE ratio confirming exposure of the target cells.Exposure to 5000 mg/kg bw triclosan did not induce a biological relevant increase in cellswith micronuclei in bone marrow cells of mice up to a sacrifice time of 72 h after treatment.

ConclusionUnder the experimental conditions used triclosan was not genotoxic (cacogenic and/oraneugenic) in bone marrow cells of mice.

Ref.: 60

Nucleus anomaly test on somatic interphase nuclei of Chinese hamsters

Study 1

Guideline: /Species: Chinese hamsters (Cricetulus griseus)

Group size: 3 rats/sex/groupTest substance: GP 41 353 (triclosan)Batch: Mg. 3Purity: /Dose level: 150, 300 and 600 mg/kg bwRoute: oral, twice 24 h apartVehicle: 0.5 % carboxymethylcellulose solutionSacrifice times: 24 h after the last dose.GLP: /Date: May 1974

Triclosan has been investigated for the occurrence of nucleus anomalies in interphase nuclei

of bone marrow cells of Chinese hamsters (Cricetulus griseus). Chinese hamsters wereexposed twice by oral gavage 24 h apart on two consecutive days. The highest dose 600mg/kg bw is approximately 1/3 of the LD50. Bone marrow cells were collected 24 h after thelast treatment. Bone marrow preparations were stained with undiluted May-Grünwald andsubsequently with 5% Giemsa and examined microscopically for nuclear anomalies. Asanomalies were registered: single Jolly bodies, fragments of nuclei in erythrocytes,micronuclei in erythroblasts, micronuclei in leucopoietic cells, bizarre forms of nuclei,polyploid cells and necrobiotic cells. Cyclophosphamide was used as positive control.

ResultsIn all triclosan exposed groups the percentage of cells displaying anomalies of nuclei did notdiffer significantly from the negative control.

Conclusion



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Under the experimental conditions used triclosan was not genotoxic in bone marrow cells ofChinese hamsters.

Ref.: 58

CommentThe test is performed before the implementations of OECD guidelines. The nuclear anomaly

test is not a routinely performed test. Purity was not reported. The test has only limitedvalue

Study 2

Guideline: /Species: Chinese hamsters (Cricetulus griseus)Group size: 6 rats/sex/groupTest substance: FAT 80 023/ABatch: 652Purity: /

Dose level: 75, 150, 300 and 600 mg/kg bwRoute: oral, thrice weekly for 12 weeksVehicle: 0.7 % carboxymethylcellulose solutionSacrifice times: 6 h after the last dose.GLP: /Date: August 1978

FAT 80023/A has been investigated for the occurrence of nucleus anomalies in interphasenuclei of bone marrow cells of Chinese hamsters (Cricetulus griseus) after long termtreatment. Chinese hamsters were exposed thrice weekly by oral gavage for 12 weeks. Thehighest dose 600 mg/kg bw is approximately 1/3 of the LD50. Bone marrow cells werecollected 6 h after the last treatment. Bone marrow preparations were stained with

undiluted May-Grünwald and subsequently with 5% Giemsa and examined microscopicallyfor nuclear anomalies. As anomalies were registered: single Jolly bodies, fragments of nucleiin erythrocytes, micronuclei in erythroblasts, micronuclei in leucopoietic cells and polyploidcells. The solvent was used as negative control; a positive control was not included.

ResultsThe bone marrow of only 3 female and 3 male hamsters (for the highest dose only 2 malehamsters) was evaluated. Of the animals treated with 600 mg/kg bw 1 died within the 2 nd week, additionally 3 by the 7th, 8th and 9th week and further 3 in the 12th week. One of theanimals treated with 300 mg/kg bw died within the 5th week. In all FAT 80023/A exposedgroups the percentage of cells displaying anomalies of nuclei did not differ significantly fromthe negative control.

ConclusionUnder the experimental conditions used FAT 80 023/A was not genotoxic in bone marrowcells of Chinese hamsters.

Ref.: 59

CommentThe test is performed before the implementations of OECD guidelines. The nuclear anomalytest is not a routinely performed test. Purity of test substance FAT 80 023/A (triclosan) wasnot reported. The test has only limited value



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Sex linked recessive lethal test in D r o s o p h i la m e l an o g a s t e r

Guideline: /Species: Male Drosophilas melanogaster of wild type strain Karsnäs 60Test substance: Irgasan

Batch no: /Purity: /Dose levels: Experiment 1: 1000 ppm Irgasan in 1% sucrose

Experiment 2: 1000 ppm Irgasan in Ringer solutionExperiment 3: 1000 ppm in corn agar substrate

Treatment: Experiment 1: In glass tubes to which 0.5 ml of the sucrose solutionwas added for 24 h

Experiment 2: Injection of 1000 ppm IrgasanExperiment 3: In ordinary vials with corn agar containing 1000 ppm

Irgasan for 7 days.GLP: not in complianceDate: 1979

Irgasan was tested in a sex linked recessive lethal assay in Drosophila melanogaster . Doseselection was based on the results of a toxicity test with adult male flies with and withoutpre-treatment with the inducer of the metabolic detoxication enzymes. Males were eithertreated with 1000 ppm Irgasan in 1% sucrose in glass tubes for 24 h, or injected with 1000ppm Irgasan in Ringer solution or kept on vials with corn agar containing 1000 ppm Irgasanfor 7 days. The flies which were treatment with Irgasan in sucrose were before treatmentput for 4 h in empty tubes in order to increase the liquid consumption. Three broods of flieswere studies: 0-3, 4-6, 7-10 days after treatment. New virgin females were given to themale for each brood.Flies treated with Irgasan in sucrose solution and flies injected with Irgasan in Ringersolution were analysed chemically by GLC for the determination of the Irgasan content

immediately after treatment and between 1 and 4 days after.

ResultsBoth in flies treated with Irgasan in sucrose solution or injected with Irgasan in Ringersolution the concentration of Irgasan reached control values in less than less 72 h or 48 h,respectively. Apparently Irgasan is excreted very fast and will thus not accumulate in theflies.No effect of Irgasan in any of the broods within the experiments was recorded. Thefrequency of the lethals recorded after treatment with Irgasan is in accordance both withthe parallel and the historic controls with this particular wild type strain.

ConclusionUnder the experimental conditions described, Irgasan did not induce an increase in sexlinked recessive lethals and, consequently, Irgasan was not genotoxic in the sex linkedrecessive lethal test in Drosophila melanogaster.

Ref.: 51

CommentThe test was not performed according the OECD guideline. Batch number and purity werenot reported. The test has only limited value.

Dominant lethal test

Guideline: /

Species: NMRI miceGroup sizes: 12 male mice/group



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Test substance: GP 41 353Solvent: 2% sodium carboxymethylcelluloseBatch: 3Purity: /Dose levels: 750 and 1500 mg/kg bwRoute: oral gavage

Sacrifice times: females were autopsied at day 14 of pregnancyGLP: /Date: October 1971

GP 41 353 has been investigated for the induction of dominant lethals in mice. Mice wereexposed by oral gavage with 750 and 1500 mg/kg bw which are equal to approximately 1/6and 1/3 of the LD50 respectively. Each male mouse from each group was mated with 4untreated females immediately after treatment. After 1 week the females were removedfrom the cages and replaced by another group of 3 untreated females. This procedure wascontinued for 8 consecutive weeks. This time of 8 “mating periods” comprises all stages ofthe maturation of the male germ cell. The mated females were sacrificed and autopsied onthe 14th day of pregnancy. The number of alive and dead foetuses as well as early

embryonic resorptions was listed. A positive control was not included.

ResultsUntil 3 days after treatment the males of the high dose group were found to display signs ofintolerance as indicated by slight convulsions. The symptoms did not exert any influence onthe mating performance. An increase in the number of implementations, live embryos,death embryos and early embryonic resorptions due to treatment with GP 41 353 was notobserved.

Conclusion:Under the conditions tested GP 41 353 did not induce an increase in dominant lethals andconsequently is not genotoxic in this dominant lethal test in mice.

Ref.: 65

CommentThe test is performed before the implementations of OECD guidelines. Purity was notreported. The test has only limited value.

Chromosome aberration test in male germinal epithelium of the mouse

Study 1

Guideline: /Species/strain: Male NMRI miceGroup size: 6 mice /groupTest substance: FAT 80 023/ABatch: 652Purity: /Dose level: 189, 378, 756 and 1512 mg/kg bwRoute: oral by gavage, on 5 consecutive daysVehicle: 2% CMCSacrifice times: 1 day after the last doseGLP: /Date: December 1978

FAT 80 023/A has been investigated for the induction of chromosome aberrations in male

germinal epithelium cells, in particular on spermatogonia, of mice. Mice were exposed on 5consecutive days by oral gavage. Germinal epithelium cells were collected 1 day after the



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last dose. Three h before section the mice were injected intraperitoneally with 10 mgcolcemid/kg. Germinal epithelium cell preparations were stained and examinedmicroscopically for chromosome aberrations. The solvent was used as negative control; apositive control was not included.

Results

In the 1512 mg/kg bw group 7 out 8 animals died in the course of the second to lasttreatment. The remaining animal was not included in the evaluation. In the other treatmentgroups all animals survived.In comparison to the vehicle control animals, FAT 80 023/A did not induce a biologicallyrelevant or statistically significant enhancement in the number of cells with chromosomeaberrations at the three remaining dose levels tested.

ConclusionUnder the experimental conditions used FAT 80 023/A is not genotoxic (clastogenic) ingerminal epithelium cells (spermatogonia) of mice.

Ref.: 61

CommentThe test is performed before the implementations of OECD guidelines. Purity of testsubstance FAT 80 023/A (triclosan) was not reported. Raw nor summarized data on theoccurrence of chromosomal aberrations were not available in the report. The test has onlylimited value.

Study 2

Guideline: /Species/strain: Male NMRI miceGroup size: 6 mice /group

Test substance: FAT 80 023/ABatch: 652Purity: /Dose level: 189, 378, 756 and 1512 mg/kg bwRoute: oral by gavage, 5 intermittently doses on days 0, 2, 3, 5 and 9Vehicle: 2% CMCSacrifice times: 3 days after the last doseGLP: /Date: February 1979

FAT 80 023/A has been investigated for the induction of chromosome aberrations in malegerminal epithelium cells, in particular on spermatogonia, of mice. Mice were exposedintermittently on days 0, 2, 3, 5 and 9 by oral gavage. Germinal epithelium cells werecollected 3 days after the last dose. Three h before section the mice were injectedintraperitoneally with 10 mg colcemid/kg. Germinal epithelium cell preparations werestained and examined microscopically for chromosome aberrations. The solvent was used asnegative control; a positive control was not included.

ResultsIn the 1512 mg/kg bw group only 4 animals survived. These animals were not included inthe evaluation. In the other treatment groups all animals survived.In comparison to the vehicle control animals, FAT 80 023/A did not induce a biologicallyrelevant or statistically significant increase in the number of cells with chromosomeaberrations at the three remaining dose levels tested.

Conclusion



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Under the experimental conditions used FAT 80 023/A is not genotoxic (clastogenic) ingerminal epithelium cells of mice.

Ref.: 62

CommentThe test is performed before the implementations of OECD guidelines. Purity of test

substance FAT 80 023/A (triclosan) was not reported. The test has only limited value.

Summary: genotoxicity/mutagenicity of triclosan in vivo

strain/cell type testcompound

concentration result quality ref

Host mediated assay with S. typhimurium in mice

albino NMRI mice FAT 80 023/A* 50-400 mg/kg bw negative limitedvalue

54

Host mediated assay with Mouse lymphoma cells in mice

DBA/2f/Bom mice FAT 80 023/A* 1313 mg/kg bw negative limitedvalue

49

Mouse spot test

T stock x C57BL/6JHan Irgasan DP 300 50 mg/kg bw mutagenic limitedvalue

63

T stock x C57BL/E Irgasan DP 300 1-25 mg/kg bw negative supportiveevidence

64

Bone marrow chromosome aberration test

Chinese hamsters(female)

GP 41 353 150-600 mg/kg bw negative limitedvalue

55

Chinese hamsters(female)

FAT 80 023/A* 75-600 mg/kg bw

long term exposure

negative limitedvalue

56

Wistar rats FAT 80’023/Q* 4000 mg/kg bw negative limitedvalue

57

Bone marrow micronucleus test

CD-1 mice triclosan 5000 mg/kg bw negative appropriate 60

Nucleus anomaly test on somatic interphase nuclei

Chinese hamsters GP 41 353 150-600 mg/kg bw negative limitedvalue

58

Chinese hamsters FAT 80 023/A* 75-600 mg/kg bw

long term exposure

negative limitedvalue

59

Sex-linked recessive lethal test

Drosophila melanogaster,strain Karsnäs 60

Irgasan 1000 ppm negative limitedvalue

51

Dominant lethal test

NMRI mice GP 41 353 750-1500 mg/kg bw negative limitedvalue

65

Chromosome aberration test in male germinal epithelium cells

male NMRI mice FAT 80 023/A* 189-1512 mg/kg bw negative limitedvalue

61

male NMRI mice FAT 80 023/A* 189-1512 mg/kg bw negative limitedvalue

62



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3.3.7. Carcinogenicity

Carcinogenicity studies of 18 months, 104 weeks, and 90 to 95 weeks in duration have beenconducted in the rat, mouse, and hamster to assess chronic exposure effects of triclosan(Pharmaco LSR, 1995 (66); Ciba-Geigy, 1986 (67); Huntingdon Life Sciences, 1999 (68)].The three life-time bioassays were designed according to OECD guidelines Nos. 451 and/or453, and GLP compliant.

Triclosan showed no tumourigenic effects in the rat and the hamster studies. The mouseoncogenicity study showed both hepatic adenomas and hepatocellular carcinomas followingan 18-month exposure to triclosan. Descriptions of the rat, mouse, and hamster data arefound in the table below.

Table 16: Findings from GLP Carcinogenicity Studies for Triclosan

Species(Strain)

DosingRegimen

Durationof

Treatment

Major Findings1 Reference;GLP and

OECDStatus

Mouse(CD-1)

Oral (diet)doses of 0,10, 30, 100,or 200mg/kg bw/d

50/sex/dose +20/sex/ dose(6-monthinterim)

18 months Decreased survival occurred in males (100 mg/kg bw/d)and high-dose females (a smaller decrease in high-dosemales was not significant); therefore, only the survivalin females was considered to be affected by triclosan.Increased plasma levels of liver function enzymes wereobserved in higher dose mice, and decreased cholesterolwas measured at all doses. There were no biochemicalor pathological indications of kidney effects. Increasedincidences of hepatic nodules or masses ordiscolorations were observed in higher dose animals.No histopathological changes other than in liver(hepatocellular hypertrophy in all animals except low-dose females) were attributed to triclosan treatment.

Data show dose-dependent increases in liver adenomasand carcinomas, with increased incidence in males vs.

females. Incidences of mice bearing at least 1 hepatictumour (i.e., combined adenoma and carcinoma data)were: 6, 10, 17, 32, 42 in males and 0, 1, 3, 6, 20 infemales at doses of 0, 10, 30, 100, and 200 mg/kgbw/d, respectively. In summary, effects in the liver,serum cholesterol decreases (males and females) andhepatocellular hypertrophy (males only) were observedat the lowest dose of 10 mg/kg bw/d; investigatorsconcluded that there was no NOEL based on liverchanges at all dose levels.

PharmacoLSR, 1995(66);

GLP-compliant


Rat(Sprague-Dawley

Oral (diet)doses of 0,12, 40, or127 mg/kgbw/d inmales and 0,17, 56, or190 mg/kgbw/d infemales60/sex/ dose+ 10/sex/dose or20/sex/control group(52-weekinterim)20/sex in

additionalhigh-dose

104 weeks Increased food intake was found in high-dose males.Mean body weight decreases were observed to betransient during the study except for consistentsignificant decreases of up to 10% in high-dose females.Sporadic changes such as slight changes in protein,glucose, bilirubin, triglyceride, and blood urea nitrogenfound in the first 52 weeks of the study had disappearedby 78-104 weeks. Slight changes in erythroidparameters were sporadic. Decreases in monocytes andwhite blood cells in F and increased clotting time inmales was observed at 104 weeks. Absolute adrenalweights were increased in mid-dose males; absolutebrain weights were decreased in high-dose males;absolute and relative ovary weights were increased inhigh-dose females; absolute and relative spleen weightswere decreased in mid-dose females, and relativespleen weights were decreased in high-dose females at104 weeks. Hepatocyte hypertrophy and hepatocytic

inclusions (hyaline-staining) were occasionally noted inmale rats at early time points but were not seen at 104

Ciba-Geigy,1986(67);

GLP-compliant




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Species(Strain)

DosingRegimen

Durationof

Treatment

Major Findings1 Reference;GLP and

OECD

Status

group (247or 422mg/kg bw/din males andfemales) at52 weeks

weeks. No other histological lesions, either neoplasticor non-neoplastic, were observed that were consideredto be treatment-related. Specifically, there were notreatment-related tumours, including hepatic tumours,in any of the treated rats examined histologically at 52or 104 weeks. Animals in the additional high-dosegroup killed at 52 weeks (247 or 422 mg/kg bw/d inmales and females, respectively) showed similar typesof toxicity as the main study animals, excepting that theseverity or incidences of events were increased. Notumours were observed in these animals. In summary,the results show that triclosan is not tumourigenic in a2-year study in rats at doses of up to 127 mg/kg bw/din males and 190 mg/kg bw/d in females. A NOEL of1,000 ppm was determined (48 mg/kg bw/d for femalesand males combined).

Hamster

(Syrian)

Oral (diet)

doses of 0,12, 75, or250 mg/kgbw/d

60/sex/ dose+ 10/sex/dose (52-weekinterim)

Females:

90 weeksMales: 95weeks

Survival was significantly decreased in males and was

generally poor (38-58%) in females at 90 weeks. Bodyweight gain was significantly decreased in all high-dosehamsters and was accompanied by a slight (3%), butsignificant decrease in food consumption in high-dosefemales. Biochemical changes observed at terminationincluded increases (<50%) in blood urea nitrogen inhigh-dose hamsters and in triglycerides in mid- andhigh-dose males. Haematological changes observedincluded slight (<15%), but significant decreases inerythroid parameters in mid- and high-dose animals,increased white blood cells in high-dose animals, andincreased lymphocytes in high-dose females attermination. Significant observations at 52 weeks(interim) were mainly renal changes in high-doseanimals. At termination, renal nephropathy wasobserved in animals of all dose groups, with increasedincidence and severity at the high-dose level. Inaddition, males showed atypical hyperplasia in thestomachs (fundic region), along with spermatozoa andgerm cell effects at the high dose. One high-dosefemale showed atypical hyperplasia in the fundic regionthat was considered to be treatment-related. High-dosefemales showed distended gastric glands and a fewtreated females of all doses showed benign papillomasof the non-glandular region of the stomach (notdiscussed in report). Hepatic effects were few, with onlyrarified hepatocytes reported in a few male animals(6/60 in high dose vs. 3/121 in controls). There wereno tumours considered to be treatment-related. Insummary, triclosan had little to no effect in hamsters at12 and 75 mg/kg bw/d, and toxic effects at 250 mg/kg

bw/d that resulted in the general deterioration of high-dose males after Week 80. Overall results show thattriclosan is not tumourigenic in hamsters at doses of upto 250 mg/kg bw/d. However, survival in females at 90weeks was poor. NOAEL=75 mg/kg bw/d.

Huntingdon

LifeSciences,1999(68);

GLP-compliant


1 Findings reported in table are significant compared to controls unless otherwise noted in text.

Doses tested in the 3 carcinogenicity assays were comparable, ranging from 10 to200 mg/kg body weight/day in mice, 12 to 127 mg/kg body weight/day (for males) and 17to 190 mg/kg body weight/day (for females) in rats, and 12 to 250 mg/kg body weight/day

in hamsters. Survival was not altered by triclosan treatment in the rat study, whereasdecreased survival was observed in both the hamster and the mouse studies. In general,neither biochemistry/clinical chemistry nor haematology analyses showed any serious



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effects that could be attributed to triclosan treatment, except in the case of liver-relatedchanges in the mouse study.

Rats showed no changes in liver function enzyme activity in plasma, including alanineaminotransferase (ALT) and aspartate aminotransferase (AST), which would be indicative ofhepatic damage. Liver effects in rats in the carcinogenicity study were limited to increased

incidences of hepatocyte hypertrophy and hyaline-staining inclusions in high-dose males atearly time points in the study. These findings were not observed in animals at thetermination of the study.

As with rats, hamsters showed no alterations in liver function enzymes. The effects oftriclosan in hamster liver, as observed in the carcinogenicity study, were limited to a slightdecrease in organ weight in high-dose females. This finding was attributed to the decreasedbody weight gain at this dose level. In addition to the organ weight effects, slight, butstatistically significant, decreases in erythroid parameters and in triglycerides weremeasured in mid-dose females. The only notable histopathological finding was an increasedincidence of rarified hepatocytes in a few high-dose males.

In contrast to observations in rats and hamsters, liver effects in the mouse includedincreased liver function enzymes in plasma; decreased blood cholesterol levels; significantincreases in liver weights; increased incidences of hepatic nodules, discolorations, ormasses; hepatocellular hypertrophy; and, increased incidences of both hepatocellularadenomas and hepatocellular carcinomas, depending on dose level. Signs of liver effects,increases in cholesterol (both sexes) and hypertrophy (males only) were seen at the lowestdose of 10 mg/kg body weight/day. Hypertrophy was seen in mice of both sexes at30 mg/kg body weight/day, and increases in liver function enzyme levels were seen in miceof both sexes at doses of 100 and 200 mg/kg body weight/day. Increases in numbers ofhepatic tumours were observed at doses of 30 mg/kg body weight/day and higher.

Summary and NOAEL Values from the Rodent Carcinogenicity Studies

Three rodent lifetime bioassays have been conducted to evaluate the carcinogenic potentialof triclosan. Triclosan produced hepatic effects and hepatic tumours in mice, but littleevidence of toxicity and no tumours in rats. Hamsters showed increased liver toxicityrelative to the rat, but no tumours.

No NOAEL could be determined for the mouse, based on findings of liver effects at all doses(effects of hepatocyte hypertrophy and decreased plasma cholesterol). Increased incidencesof liver tumours were observed at doses of 30 mg/kg body weight/day and higher in mice.

It should be noted that triclosan is a peroxisome proliferator in mouse liver.

The NOEL for the rat study was determined by study investigators to be the mid-dose of1,000 ppm, or ~48 mg/kg body weight/day for males and females combined, based on thefinding of hepatocyte changes in the high-dose group of 3,000 ppm (~127 mg/kg bodyweight/day in males and 190 mg/kg body weight/day in females). Although absoluteadrenal weights were elevated at the 1,000 ppm dose in rats, this change was notconsidered to be adverse for a number of reasons, including a lack of change in relativeadrenal weights, a lack of change in adrenal weights in the high-dose group, and theabsence of accompanying histopathological changes in the adrenal glands.

The NOAEL for the hamster study was determined to be the mid-dose of 75 mg/kg bodyweight/day. Although the values for certain haematologic parameters were significantlyaltered at doses of 12 and 75 mg/kg body weight/day, the erythroid changes were slight(on the order of 5 to 10%), and not considered to be adverse effects per se. An increase intriglycerides (40 to 50%) was not considered to be an adverse effect, as the increase was

not accompanied by any changes in liver weight or histopathology.The NOAEL values for the three rodent lifetime cancer bioassays are summarized in Table17.



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Table 17: Summary of NOAEL Values from GLP Carcinogenicity Studies for Triclosan inRodents

Species NOAEL (mg/kg bw/day) Comment

Mouse 10(this value is a LOEL for tumour

formation only)

No overall NOAEL due to hepatotoxic effectsobserved at the lowest dose tested. There were no

tumours observed in other tissues.

Rat ~48 (NOAEL)1 NOAEL based on systemic toxicity. There was noevidence of tumour formation, including in liver.There was no evidence of hepatotoxicity.

Hamster 75 NOAEL based on systemic toxicity. There was noevidence of tumour formation, including in liver.

1 1,000 ppm ~48 mg/kg body weight/day for males and females, combined.

CommentAccording to the EU classification system, triclosan is not considered classifiable as acarcinogen. It should be noted that triclosan is a peroxisome proliferator in mouse liver.

3.3.8. Reproductive toxicity

The reproductive and developmental toxicology of triclosan has been investigated interatology studies in the mouse, rat, and rabbit, and a two-generation reproductive toxicitystudy in the rat. Based on international guidelines (ICH, 2005), effects on fertility andreproduction and perinatal development should be evaluated in rodents (typically, the rat),and teratology studies be conducted in both rodent and non-rodent species (typically, therat and the rabbit). The available studies for triclosan meet these recommendations.

3.3.8.1. Two generation reproduction toxicity

One GLP-compliant two-generation study was conducted in rats, providing both fertility andreproduction and post-natal data [Morseth, 1988 (69)]. The study was consistent withOECD guidelines (Two-Generation Reproductive Toxicity Study), with the exception that thecoagulating gland was not preserved, and with ICH (Fertility and Early EmbryonicDevelopment, Stages A and B; Pre- and Post-Natal Development, ICH Stages D, E, and F)guidelines, with the exception that sperm counts and viability assessments, and specifictests for physical, sensory, reflexes and behaviour, were not conducted. In addition to thepost-natal data provided in the two-generation rat study, the GLP-quality study in ColworthWistar rats provided post-natal data [Denning et al ., 1992 (74)]. This study was generallyconsistent with international guidelines, with the major exceptions being those listed above[i.e., as for the study by Morseth, 1988 (69)], that maturation and fertility were notassessed, and that relatively few animals (5 per treatment group) were allowed to deliverand rear offspring. The fertility, reproduction, and post-natal data from these studies are

summarized in the table below.

Table 18: Findings from the Reproductive and Developmental Toxicity Studies thatExamined Fertility and Reproduction and/or Post-Natal Parameters

Species

(Strain)

Dosing

Regimen(mg/kg bw/d)

Major Findings1 Reference,

GLP andOECD

Status

CharlesRiverCD® (SD)Br

rats

0, 300, 1,000, or3,000 ppm(Doses of ~17,56, and 176

mg/kg bw/d inmales and ~23,

Fertility and Reproduction:F0 Generation: There were no remarkable effects on F0 treated ratsat any dose, including no effects on mating, pregnancy, duration ofgestation, and parturition.

F1 Generation: There were no treatment-related effects onreproduction indices.

Morseth,1988(69)

GLP-compliant



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Species(Strain)

DosingRegimen

(mg/kg bw/d)

Major Findings1 Reference,GLP and

OECD

Status

73, and 229mg/kg bw/d infemales, basedon foodconsumption andbody weight dataafter 10 weeks ofdosing.

Both males andfemales weredosed starting 10weeks prior tomating. The ratsreceived theappropriatecontrol or testdiet throughout

the growth,mating,gestation, andlactation phases,or until terminalsacrifice.

Post-natal Parameters:

F1 Generation: Post-natal survival from Day 0 to Day 4 (i.e.,

viability index) was slightly reduced in the high-dose group (90,94, 96, and 82% in the control, 300, 1,000, and 3,000 ppmgroups, respectively). Adjusted mean body weights of pups in thehigh-dose group were generally lower than controls throughout theDay 0 to 21 lactation period. With regard to development, meanbody weights of high-dose pups were lower than controls duringWeeks 0 to 12. Mean body weights were slightly decreased in low-dose group females vs. controls during Weeks 4-8 and 11. Overall,the most common findings were dilated pelvises of the kidney thatshowed a slight increase in incidence at the high dose in pups, butno differences between treatment groups in adult animals.F2 Generation:The mean number of live F2 pups on Day 0, viability index (survivalto Day 4), and survival to weaning were slightly lower in high-doseanimals vs. controls. Adjusted mean body weights of high-dose

pups were slightly, but significantly lower than controls on Day 0.There were no other remarkable treatment-related findings in theF2 pups, or of mature F2 offspring.

Although study investigators considered the data to be somewhatequivocal, they determined the Overall NOAEL for the study to be1,000 ppm (~65 mg/kg bw/d for males and females, combined),based on evidence of slightly reduced survival and pup bodyweights at the high dose. However, the data in the study indicatethat a Fertility and Reproduction NOEL of 3,000 ppm (~203mg/kg bw/day for males and females combined) would beappropriate based on the absence of treatment-related effect inboth the F0 and F1 generations. The Foetal and Post-Natal

NOAEL would be 65 mg/kg bw/day (based on pup body weights).


Rat(ColworthWistar)

Dams received 0,30, 100, 300mg/kg bw/day)via oral gavage,in corn oil onDays 6-15 ofgestation

[Maternal and Foetal Data are presented in Table 19]Post-natal Parameters: Survival and development of pups frombirth to weaning was comparable to controls. There was nosignificant increase in the number of pups with anomalies.

Study investigators did not determine NOEL values for this study.However, the data indicate that a Foetal and Post-Natal NOEL of300 mg/kg bw/d would be appropriate, based on the lack of foetaland pup effects at this dose.

Denning et

al ., 1992(70);

GLP-compliant2

OECD:comparable

1 Statistically and biologically significant findings have been outlined.2 Based on the presence of a statement of Quality Assurance, the study by Denning et al ., [1992 (74)] in ColworthWistar rats was assumed to contain GLP-quality data.

3.3.8.1.1 Fertility and Reproduction

Fertility and reproduction parameters of mating, pregnancy, duration of gestation, andparturition were assessed in the two-generation rat study [Morseth, 1988 (69)]. Based onthe absence of effects in F0 and F1 generation rats, the NOAEL (NOEL) for fertility andreproduction was determined to be 3,000 ppm (~203 mg/kg body weight/day using maleand female doses, combined), the highest dose tested.

3.3.8.1.2 Post-Natal Parameters

In the two-generation study that examined post-natal development in rats, the primaryfindings were a slight decrease in mean foetal body weights and a slight decrease in theDay 0 to Day 4 survival in the F1 and F2 generations at the high dose of 3,000 ppm(~203 mg/kg body weight/day) [Morseth, 1988 (69)]. Thus, the foetal and post-natalNOEL for this study was determined to be 1,000 ppm (~65 mg/kg body weight/day using



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combined male and female doses) for triclosan administered in the diet. Results from thestudy in Colworth Wistar rats showed no effect of triclosan on the survival and developmentof pups from birth to weaning [Denning et al ., 1992 (70)]. In this study, the post-natalNOEL was determined to be 300 mg/kg body weight/day for triclosan administered via oralgavage.

In summary, taking into account both of the studies containing post-natal data, the lowestpost-natal NOAEL is considered to be 65 mg/kg body weight/day, the post-natal NOEL fromthe two-generation study using dietary administration of triclosan.

3.3.8.2. Teratogenicity

One-Generation Reproduction Toxicology: Developmental Toxicity Studies

3.3.8.2.1 GLP Studies

GLP-compliant teratology studies were conducted in mice, rats, and in rabbits using the oralroute of administration. All 3 of the main studies were conducted along OECD guidelines.

One preliminary range-finding study was conducted for each species, in addition to thedefinitive investigations. NOEL or NOAEL values were determined for each of the definitivestudies in each species. Brief summaries of the pertinent findings from the GLP teratologystudies are presented in table 19. Where appropriate, findings of foetal effects wereclassified either as foetal variations (an alteration that may occur at a relatively highfrequency and/or represent a (reversible) retardation or acceleration in development, atransitory alteration, or a permanent alteration not considered to adversely affect survival,growth, development, or functional competence in a given species or strain) or foetalmalformations (permanent change/anomalies in which there is a morphologic defect of anorgan, resulting from an abnormal developmental process that occur at low incidences in agiven species or strain of animal) (U.S. FDA, 2001).

Table 19: Findings from GLP Teratology/Developmental Toxicity (Segment 2, ICH Stage C)Studies for Triclosan

Species(Strain)

DosingRegimen

(mg/kg

bw/d)

Major Findings1 Reference,GLP and OECD

Status

Mouse(Crl:CD®-1(ICR)BR)

0, 5, 10, 20,40, 80, or 160mg/kg bw/d via the diet(Days 6-15 ofgestation)

Dose range-finding study.Maternal Parameters: Maternal body weight gain andfood consumption were reduced in the 160 mg/kg bw/dgroup. At the doses of 80 and 160 mg/kg bw/d, absoluteliver weights and relative liver weights (relative toterminal body weights and to brain weights) wereincreased.Foetal Parameters: Foetal body weight data were lowerfor the 40, 80, and 160 mg/kg bw/d groups than for thevehicle control group. Litter averages for resorptions(early and late resorptions, percentage of resorbedconceptuses and the number of dams with resorptions)were increased at 160 mg/kg bw/day. There were noother remarkable findings.

Study investigators did not determine NOEL values for thisstudy.

Argus ResearchLaboratories,1992a(70)

GLP-compliant

OECD: notapplicable for adose rangefinding study

Mouse(Crl:CD®-1(ICR)BR)

0, 10, 25, 75,or 350 mg/kgbw/d in thediet(Days 6-15 ofgestation)

Maternal Parameters: Body weights and body weightgains were increased in the 350 mg/kg bw/d group.Absolute liver weights and relative liver weights (relativeto terminal body weights and to brain weights) weresignificantly increased in the 75 and 350 mg/kg bw/d dosegroups. The 350 mg/kg bw/d dose caused increases in thenumbers of mice with tan-coloured livers, along with one

Argus ResearchLaboratories,1992b(71)

GLP-compliant



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Species(Strain)

DosingRegimen

(mg/kg

bw/d)


Status

mouse in the 75 mg/kg bw/d dose group.Foetal Parameters: Foetal body weight data wereslightly lower for the 75 and 350 mg/kg bw/d groupscompared to the vehicle control group. There werereversible delays in ossification caused by the test articlein the 75 and 350 mg/kg bw/d dosage groups, includingskull ossification and reductions in the average numbersof ossified forepaw phalanges and hind paw phalanges.There were no other remarkable findings.

Maternal NOEL = 25 mg/kg bw/d (based on tan-coloured livers and increased liver weights)Foetal NOEL = 25 mg/kg bw/d (based on decreasedfoetal body weights and delayed ossification)

OECD:consistent with “Teratogenicity”guideline

Rat(Charles RiverCD® Sprague-

Dawley derived)

0, 5, 10, 25,50, or 75mg/kg bw/d via

oral gavage, in1%carboxymethyl-cellulose in a20% glycerinein watersuspension(Days 6-15 ofgestation)

Dose range finding study.Maternal Parameters: The lower maternal bodyweights that occurred at the high dose were due to the

weight loss in a single dam. There were no otherremarkable findings.Foetal Parameters: Foetal body weight data were lowerfor all treatment groups vs. controls; however, only the75 mg/kg bw/d group had foetal body weights outside thelow range of historical control data. There were no otherremarkable findings.


Bio/dynamics,1992a(72)

GLP-compliant

OECD: notapplicable for adose rangefinding study

Rat(Charles RiverCD® Sprague-Dawley derived)

0, 15, 50, or150 mg/kgbw/d via oralgavage, in 1%carboxymethyl-

cellulose in a20% glycerinein watersuspension(Days 6-15 ofgestation)

Maternal Parameters: There was a slight but significantdecrease in food consumption from Days 6 through 11 ofgestation at the high dose (70±7 vs. 76±5 g/kg bw/d incontrols). There were no other remarkable findings.Foetal Parameters: Foetal development showed

retarded ossification at the high dose (cranium, vertebrae,sternebrae, metacarpals, and pelvic girdle). There wereno other remarkable findings.

Maternal NOEL = 50 mg/kg bw/d (based on decreasedfood consumption at the high dose)Foetal NOEL = 50 mg/kg bw/d (based on delayedossification at the high dose)

Bio/dynamics,1992b(73)

GLP-compliant


Rat (ColworthWistar)

0, 30, 100, 300mg/kg bw/d via

oral gavage incorn oil(Days 6-15 ofgestation)

Maternal Parameters: At the 300 mg/kg bw/d doselevel, slight maternal toxicity was manifested as transientdiarrhoea, retarded body weight gain (e.g., 9.4 vs. 13.0 gfor controls during the period of Days 6 to 10), andreduced food consumption (5-15% decrease vs. controls),and increased water intake (<10% increase vs. controls).There were no other remarkable findings.

Foetal Parameters: There were no remarkable findings.

Study investigators did not determine NOEL values for thisstudy. However, the data indicate that a Maternal NOEL of 100 mg/kg bw/d would be appropriate, based ondecreased body weights and slight diarrhoea, with aFoetal NOEL of 300 mg/kg bw/d (based on a lack offoetal effects).

Denning et al .,1992(74);

GLP-compliant2

OECD:

comparable

Rabbit(New ZealandWhite)

0, 5, 10, 25,50, or 75mg/kg bw/d viaoral gavage in1%carboxymethyl-cellulose in a20% glycerinein water

Dose range-finding study.Maternal Parameters: There were slight meanmaternal body weight losses and lower terminal bodyweights of the does treated with 75 mg/kg bw/d, as wellas decreased food consumption as measured on Days 6,8-11, 13, and 16, but not on Days 7 and 18, for this dosegroup. There were no other remarkable findings.Foetal Parameters: There were no remarkable findings

Bio/dynamics,1992c(77)

GLP-compliant

OECD: notapplicable for adose range



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Species(Strain)

DosingRegimen

(mg/kg

bw/d)


Status

suspension(Days 6 to 18of gestation)


finding study

Rabbit(New ZealandWhite)

0, 15, 50, or150 mg/kgbw/d via oralgavage in 1%carboxymethyl-cellulose in a20% glycerinein watersuspension(Days 6-18 ofgestation)

Maternal Parameters: There were slight decreases inmean maternal body weight (-5.1% at Day 10 to -7.9% atDay 16, with significant decreases on Days 14 and 16)and occasional decreased body weight gains during thedosing period at the high dose, as well as decreased foodconsumption for this dose group (from -7.05 at Day 11 to-41.4% at Day 14). There were no other remarkablefindings.Foetal Parameters: There were no remarkable findingsat any of the doses tested.

Maternal NOEL = 50 mg/kg bw/d (based on decreasedmaternal body weights and food consumption)Foetal NOEL = 150 mg/kg bw/d (based on the absence

of effects at the highest dose tested)

Bio/dynamics,1992d(78)

GLP-compliant


1 Being consistent with industry standards, statistical analyses were conducted for definitive, but not range-finding,studies. Statistically and biologically significant findings have been outlined.2 Based on the presence of a statement of Quality Assurance, the study by Denning et al . [1992 (74)] in ColworthWistar rats was assumed to contain GLP-quality data.

GLP Studies in the Mouse

The potential for triclosan to induce developmental toxicity effects has been investigated in1 range-finding and 1 definitive teratology study in mice [Argus Research Laboratories,1992a (71); Argus Research Laboratories, 1992b (72)]. Based on the study report, thedefinitive study was consistent with OECD (Teratogenicity) and ICH (Embryo-foetaldevelopment, Stage C) guidelines, with the exception that the placenta was not grosslyexamined. This deviation would not have affected the interpretation of the results. The test

article was administered via the diet; as triclosan did not affect food consumption, thecalculated average dosages were comparable to the targeted dose levels in the definitivestudy.

Doses in the definitive mouse study were 0, 10, 25, 75, or 350 mg/kg body weight/dayadministered in the diet on Days 6 to 15 of gestation. Maternal toxicity was observed atdoses greater than 25 mg/kg body weight/day, including liver effects (i.e., increased liverweights and tan-coloured livers). Triclosan was not teratogenic in either the dose rangefinding or the definitive study. Foetal effects (classified as foetal variations) includedslightly decreased body weights at the two higher doses that also caused maternal toxicity,as well as reversible delays in ossification at the same doses. A foetal NOEL of 25 mg/kgbody weight/day was determined based upon decreases in foetal body weights and delayed

ossification at higher dose levels in the definitive study.

GLP Studies in the Rat

In rats, triclosan has been investigated in 1 range-finding and 2 definitive teratology studies[Bio/dynamics, 1992a (73); Bio/dynamics, 1992b (74); Denning et al ., 1992 (70)]. Thestudy in Colworth Wistar rats [Denning et al ., 1992 (70)], although lacking a formalstatement of GLP compliance, included a statement of Quality Assurance and was,therefore, considered to contain GLP-quality data. Based on the reports, the studies weregenerally consistent with the OECD (Teratogenicity) and ICH (Embryo-foetal development,Stage C) guidelines, with the major exceptions being that the placenta was not grosslyexamined in the Bio/dynamics studies, and there were relatively few numbers of animals inthe post-natal survival and development portion of the study by Denning et al . (see Section

3.8.1). Triclosan was orally administered by gavage in all of the studies, although differentvehicles were used (1% carboxymethylcellulose in 20% glycerine in water suspension in the



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Bio/dynamics studies, and corn oil in the Colworth Wistar rat study). Rats were treated onDays 6 to 15 in each of the studies.

Doses of 0, 15, 50, or 150 mg/kg body weight/day were administered in the definitive oralgavage study using triclosan in 1% carboxymethylcellulose [Bio/dynamics, 1992b (73)].The dams showed evidence of maternal toxicity in the form of slight, but significant,

decreases in food consumption from Days 6 to 11 of gestation in high-dose animals. Therewas no evidence of any teratogenic effect of triclosan. Foetal effects (foetal variations)were manifest as delayed ossification at the high dose of 150 mg/kg body weight/day, andwith maternal toxicity occurring at the same dose. Based on observations of delayedossification at the high dose, the foetal NOEL in this study was determined to be 50 mg/kgbody weight/day. The maternal NOEL was also 50 mg/kg body weight/day.

The 1992 study by Denning et al . was conducted in Colworth Wistar rats at doses of 0, 30,100, or 300 mg triclosan/kg body weight/day administered via oral gavage in corn oil, andexamined both embryo/foetal and post-natal parameters. The results from this studydemonstrated no foetal effects in terms of anomalies, body weights, numbers of livefoetuses, etc . Maternal toxicity was in the form of delayed body weight gain, and reduced

food consumption, as well as transient diarrhoea, at the highest dose, resulting in amaternal NOEL of 100 mg/kg body weight/day. Based on the absence of foetal effects atany of the doses tested, the foetal NOEL for this study was determined to be 300 mg/kgbody weight/day.

In summary, triclosan showed no teratogenic effects at any of the doses in either of the GLPstudies in rats. The only embryo or foetal toxic effect observed was classified as a foetalvariation (delayed ossification) in the 1% carboxymethylcellulose vehicle study at the doseof 150 mg/kg body weight/day, a dose that was associated with evidence of maternaltoxicity (significantly decreased food consumption). The findings of maternal toxicity at thehigh dose in each of the studies indicated that dose levels were adequately high in eachcase. (It should be noted that the observed decreases in food consumption followed the

administration of triclosan by oral gavage, and therefore would not likely have been due toany decreased palatability of the diet.) Hepatotoxic effects were not noted in the dams inany of the studies. No embryo or foetal toxic effects were seen at any of the doses in thecorn oil study. None of the studies showed evidence of any teratogenic effects of triclosan.Taking into account both of the GLP rat teratology studies, the foetal NOEL is considered tobe 50 mg/kg body weight/day in rats given triclosan via oral gavage in a 1%carboxymethylcellulose vehicle, with the maternal NOEL being also 50 mg/kg bodyweight/day.

GLP Studies in the Rabbit

In rabbits, triclosan has been investigated in one range-finding and one definitive teratologystudy [Bio/dynamics, 1992c (75), Bio/dynamics, 1992d (76)]. Based on the reports, the

studies were generally consistent with the OECD (Teratogenicity) and ICH (Embryo-foetaldevelopment, Stage C) guidelines, with the major exception being that the placenta was notgrossly examined. Triclosan was orally administered by gavage in a vehicle of 1%carboxymethylcellulose in 20% glycerine in water suspension. The does were treated fromDays 6 to 18 of gestation.

Doses of 0, 15, 50, or 150 mg/kg body weight/day were administered in the definitive oralgavage study in rabbits [Bio/dynamics, 1992d (76)]. The does showed evidence ofsignificant maternal toxicity in the form of decreased body weights, body weight gains, andfood consumption at the high dose. There was no evidence of embryo or foetal toxicity orof teratogenic effect of triclosan. Based on the lack of foetal effects in this study, the foetalNOEL was determined to be 150 mg/kg body weight/day administered by gavage, with thematernal NOEL being 50 mg/kg body weight/day.



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In summary, triclosan showed no teratogenic and no embryotoxic effects at any of thedoses in the definitive GLP study conducted in rabbits. Findings of maternal toxicity(decreases in maternal body weight gains) at the high dose indicated that dose levels wereadequately high in the study. Hepatotoxic effects were not observed in any of the dams.The foetal NOEL is considered to be 150 mg/kg body weight/day in rabbits given triclosanvia oral gavage, with the maternal NOEL being 50 mg/kg body weight/day.

Non-GLP Studies

Table 20 presents summaries of the non-GLP developmental toxicity studies for triclosan,including findings from a genotoxicity study in mice that examined embryo/foetal toxicityfollowing triclosan administered via the intraperitoneal route of administration.

Table 20: Findings from non-GLP Teratology/Developmental Toxicity (Segment 2, ICHStage C) Studies for Triclosan

Species

(Strain)

Dosing

Regimen

(mg/kgbw/d)

Major Findings1 Reference,

GLP and

OECD Status

Mouse(C57Bl/E cross,specific for thegenotoxicityassayconducted)

Singleintraperitonealinjections of 0,1, 2, 4, 8, or 25mg/kg bw oneither Day 9 orDay 10 orgestation

Note that this was primarily a genotoxicity study (not areproductive/developmental toxicity study).Maternal Parameters: Only mortality was assessed.There were deaths in the high-dose group (12/41 dams),but no deaths in the 267 animals treated at the 1, 2, 4,and 8 mg/kg doses.Foetal Parameters: The principle endpoints assessedwere embryo and post-natal survival. Embryo survival(reductions in litter size) was reduced at the high dose inanimals dosed on either Day 9 or Day 10. Post-natalsurvival was markedly reduced at the high dose. Post-natal survival was slightly decreased at the dose of 8mg/kg bw (Day 9 or Day 10 treatment), and at the doses

of 2 and 4 combined.

Study investigators did not determine NOEL values for thisstudy. It should be noted that, due to the design of thestudy (e.g., lack of evaluation of parameters typicallyassessed in reproductive toxicity studies), it was notpossible to reach any clear interpretation and conclusion ofthe impact of these data.

Russell andMontgomery,1980 (64)

GLP: notspecified

OECD: notspecified

Rat(Wistar)

0, 100, 200, or400 mg/kgbw/d via oralgavage in oliveoil(Days 7-17 ofgestation)

Maternal Parameters: Clinical signs of piloerection,incontinence, and diarrhoea were observed at the highdose, together with a decrease in food consumption duringthe dosing period.Foetal Parameters: There was a significant increase innumbers of foetal deaths at the high dose of 400 mg/kgbw/d (7.05% mortality vs. 1.59% in the controls).

Study investigators did not determine NOEL values for thisstudy. However, the data indicate that Maternal andFoetal NOELs of 200 mg/kg bw/d would be appropriate,based on the absence of any significant maternal andfoetal effects at this dose level.

Kawashima et

al ., 1987 (77)

GLP: notspecified

OECD: not

specified



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Species(Strain)

DosingRegimen

(mg/kg

bw/d)

Major Findings1 Reference,GLP and

OECD Status

Rat (Wistar) andHamster(Syrian)

0, or 1/1000,1/500, 1/250,1/100, or 1/50of the LD50 dose in a pilotstudy(approximately4, 8, 16, 40,and 80 mg/kgbw/d based onthe LD50 of 4g/kg bw in rats– doses inhamsters weresimilarly1/1000, 1/500,1/250, 1/100,or 1/50 of the

LD50 dose).Animals weretreated duringgestationalDays 6-15(rats) or Days6-10(hamsters).

Limited numbers and types of parameters were assessed(dams: body weight, clinical signs, mortality; foetuses:body weights, placental weights, foetal length, foetal taillength, number of foetuses, number of males, numbers ofresorptions, gross abnormalities).Maternal Parameters (Rat): There were no remarkablefindings.Foetal Parameters (Rat): A significant decrease innumber of live foetuses and number of males was reportedin the 1/1000 (lowest dose) group vs. controls. However,this was not considered to be treatment-related based on alack of dose relationship. Also, the decrease in number offoetuses did not appear to be due to an increase inresorptions but could have reflected a decrease inovulation or implantation rate (could not be confirmed dueto lack of corpora lutea data).Maternal Parameters (Hamster): Mortality wasreported at the highest dose tested (no details provided).Foetal Parameters (Hamster): Body weights weresignificantly decreased in the high-dose group. Thesignificant decrease in number of live foetuses in the lowdose group is not considered to be treatment-related dueto the lack of a dose relationship. Also, there was noincrease in number of resorptions, suggesting that theremight have been a decrease in ovulation or implantationrate (could not be confirmed due to lack of corpora luteadata). The decrease in number of live foetuses in the high-dose group was accompanied by maternal mortality.

Study investigators did not determine NOEL values for thisstudy. It should be noted that, due to the design of thestudy (e.g., lack of evaluation of parameters typicallyassessed in reproductive toxicity studies), it was not

possible to reach any clear conclusion of the impact ofthese data.

Piekacz, 1978(78)


1 Statistically and biologically significant findings have been outlined.

Non-GLP Studies in the Mouse

In addition to the GLP studies in mice, limited reproductive toxicity data were found in apublished, non-GLP mouse “spot test” genotoxicity study [Russell and Montgomery, 1980(64)]. Since this was a genotoxicity study, and not a reproductive toxicity study, therewere a large number of deviations from international guidelines for the conduct ofreproductive toxicity studies: the lack of evaluation of typical parameters for evaluation inreproductive and developmental toxicity studies (e.g., external and internal examination of

the foetuses, litter parameters); the dosing regimen (a single injection on gestational Days9 or 10 vs. dosing throughout the period of organogenesis); the route of administration (theintraperitoneal route is not appropriate for reproductive toxicity testing); the choice ofvehicle solution (although vehicle controls were included and no toxic vehicle effects werereported, 60% methanol is not considered to be an appropriate, non-toxic vehicle for use ina reproductive toxicity study), and the choice of statistical analytical methods (it ispresumed, based on the use of the Fisher’s Exact test for the genotoxicity data, that thesame test was used for the reproductive effects data). As such, the significance of thestudy’s stated statistically-significant findings are questionable. As maternal deaths werereported to have occurred in the high-dose group, developmental toxicity findings at thisdose level could be attributed to maternal toxicity, and not to the direct toxicity of triclosanto foetuses per se. Without data from the evaluation of appropriate reproductive toxicitystudy parameters, it was not possible to reach a conclusion regarding the significance, ifany, of the post-natal survival data. Overall, the choice of methods used indicates that thedata in this non-GLP mouse study are of limited value.



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Non-GLP Studies in the Rat and Hamster

Additional reproductive and developmental toxicity data in rats were provided in two non-GLP studies [Kawashima et al ., 1987 (77); Piekacz, 1978 (78)].

Although no statement of GLP compliance was included in the published report, the study byKawashima et al . [1987 (77)] appears to have been well-conducted using methodology

comparable to published guidelines for the conduct of reproductive toxicity studies. Effectsof significantly increased foetal mortality were reported at the high dose of 400 mg/kg bodyweight/day (7.05 vs. 1.59% mortality in controls, with the accompanying finding ofincreased numbers of resorptions at the high dose)1. These effects could not conclusivelybe attributed to the direct action of triclosan, as this dose also caused maternal toxicity(diarrhoea, incontinence, piloerection, decreased food consumption). There was noevidence of teratogenic effects caused by triclosan at any of the doses tested. Althoughstudy investigators did not determine a NOEL for this study, a NOEL value of 200 mg/kgbody weight/day may be considered appropriate based on the absence of any significantmaternal or foetal effects at this dose.

The early study by Piekacz [1978 (78)] included data from experiments conducted usingboth rats and hamsters. This study was not consistent with current guidelines for the

conduct of reproductive toxicity studies, especially with regard to the parameters evaluated,the species used (i.e., hamsters are not conventionally used as they are considered to be ahighly sensitive species for reproductive studies), the number of animals (i.e., only 10 pergroup), and the statistical methods used (no evidence that a necessary correction(adjustment) for multiple comparisons was included). As maternal toxicity was notevaluated, excepting mortality and body weight changes, the doses selected for the studycould not be evaluated for appropriateness and, moreover, any reproductive ordevelopmental toxicity effects observed could not be evaluated with reference to maternaleffects. In this study, decreases in numbers of live foetuses were reported to occur at thelow dose in rats and in both the low and high doses in hamsters. These effects in the low-dose groups were not considered to be directly treatment-related, as, with regard to bothrats and hamsters, there was no dose-relationship. There was also no high-dose effect in

the rats. For the low dose groups (rat and hamster), there were no increases inresorptions, indicating that the decreased number of live foetuses may have reflecteddecreases in implantation or ovulation rates (could not be confirmed due to lack of corporalutea data). Foetal effects in hamsters of decreased foetal numbers at the high dose of 80mg/kg body weight/day were accompanied by maternal toxicity effects consisting of deaths,and thus were not clearly a direct effect of triclosan. Overall, regardless of the contentionthat the study may have been inadequately conducted compared to current standards, thestudy provides little to no evidence of any treatment-related effect of triclosan on thedevelopment of rats or hamsters. As with the study by Kawashima et al . [1987 (77)], therewere reported to be no teratogenic effects of triclosan at any of the doses tested. No NOELlevel was determined for this study.

3.3.8.3 Summary and NOAEL Values from Reproductive and Developmental ToxicologyStudies

Both GLP and non-GLP reproductive and developmental toxicology studies have investigatedthe effects of triclosan on fertility, development, parturition and lactation. The pivotalstudies were conducted pursuant to GLP regulations and generally followed the relevant ICHand OECD guidelines. Appropriate test species (i.e., rats and rabbits) and a clinicallyrelevant route of administration (i.e., oral) were used in the studies. Although toxicokineticparameters were not measured in these studies, the doses achieved in these test systemsreached as high as 300 mg/kg body weight/day in rats and 350 mg/kg body weight/day in

1 It should be noted that the mortality rates were 1.51 and 8.19% in the control and high-dose groups,respectively, as presented in the published paper by Kawashima et al . (1987). However, a re-calculation ofthe data (number of dead implants / total number of implants x 100) yielded mortality rates of 1.59 and7.05% in the control and high-dose groups, respectively. Following appropriate chi-square statistical analysis,the high dose group data were found to be significantly different from control (p<0.05).



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mice (not a species typically used in reproductive toxicity studies, due to the high sensitivityof the species) and are considered adequate to assess the potential for triclosan to causehuman reproductive toxicity. In addition, evidence of maternal toxicity was observed in allof the studies, indicating that adequately high doses of triclosan have been tested. Anoverall, integrated view of the foetal data together with maternal data is necessary whenassessing the relevance of the findings in these reproductive toxicity studies (U.S. FDA,

2001).

Taking the studies all together, there was no evidence of teratogenic effects of triclosan inany of the studies, both GLP and non-GLP, and at any of the dose levels tested in rats andrabbits. Similarly, there was no evidence of teratogenic effects in studies conducted in miceand hamsters. Furthermore, in the definitive GLP studies, the effects on developingfoetuses were limited to foetal variation effects of reversible, delayed ossification in rats,there being no effects observed in rabbits.

It is important to note that foetal variations were observed at doses that also producedsignificant maternal toxicity. In the GLP-compliant mouse study, consistent with thefindings from the rat studies, there were clear indications that the foetal variations of

significantly decreased body weight and delayed ossification observed at the two higherdoses in the mouse study were likely to be secondary to maternal toxicity consisting ofhepatic effects of tan-coloured livers and increased absolute and relative liver weights(there were no foetal effects at doses that were not maternally toxic in mice). No livereffects were observed in any of the studies in rat or rabbits using doses up to 300 mg/kgbody weight/day. While there were reports of decreases in numbers of live foetuses in thenon-GLP studies [Kawashima et al ., 1987 (77); Russell and Montgomery, 1980 (64);Piekacz, 1978 (78)], these effects also were observed at doses of triclosan that producedsignificant maternal toxicity (e.g., deaths). Thus, the reported effects were unlikely to havebeen a direct effect of triclosan but, rather, secondary to maternal toxicity. It is consideredthat only findings potentially indicative of reproductive toxicity at doses that do not producematernal toxicity are of increased concern for human reproductive or developmental

toxicity. Thus, taken altogether, triclosan was not teratogenic and provided no clearevidence of direct effects on reproduction, embryo/foetal toxicity, or post-nataldevelopmental toxicity in any of the reproductive and developmental toxicology studies.

Triclosan also has been investigated for potential actions as an oestrogen disruptor, as itschemical structure resembles that of known non-steroidal estrogens (e.g., DES, bis-phenolA). In a published, non-GLP study [Foran et al ., 2000 (79)], Japanese medaka fry wereexposed to concentrations of up to 100 µg triclosan/µL for 14 days, but there was noevidence of any effect of triclosan on sex ratios in developing fish. The results of thisexperiment were consistent with the findings from GLP studies in mammals indicating thattriclosan has no effect on sex ratios or on reproductive maturity.

NOAEL (NOEL) values from the definitive GLP studies are summarized in Table 21. Thedevelopmental toxicity effects of decreased foetal body weights and delayed ossification inall of the studies were observed at doses that also caused maternal toxicity. Based on thedata in Table 21, the mouse NOAEL (both maternal and foetal) could represent an overallNOAEL for reproductive and developmental toxicity effects of triclosan. However, asobserved in the repeated dose studies, the mouse is uniquely sensitive, showing livereffects at low doses of triclosan, including in the dams in the teratology study. Thus the lowNOAEL value for foetal effects that was determined based on the mouse study may beattributed to the sensitivity of the maternal mice to liver effects, and is not due to any directeffect of triclosan on foetuses per se. The next lowest value for an overall NOAEL forreproductive and developmental toxicity is 50 mg/kg body weight/day from the study inrats. Of note, a lack of liver effects at doses up to 300 mg/kg body weight/day was seen instudies in both rats and rabbits, indicating a consistency between these two species.



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Table 21: Summary of NOAEL1 Values from GLP Reproductive and Developmental ToxicityStudies for Triclosan

Species Route ofAdministration

MaternalNOAEL

(NOEL)1

(mg/kgbw/d)

Foetal NOAEL(NOEL)1

(mg/kg

bw/d)

Comment

Fertility and Reproduction Effects

Rat Diet 2032 No adverse effects on fertility andreproduction, based on lack of remarkablefindings in F0 and F1 pups.

Foetal Effects

Mouse Diet 25 25 Maternal liver effects noted in 2 higherdose groups. Foetal effects limited todecreased foetal body weights and delayedossification at doses that caused maternaltoxicity.

Rat Oral (gavage;

carboxymethyl-cellulose)

50 50 Decreases in food consumption in high-

dose dams. Reversible, delayedossification in foetuses at same dose.

Rat Oral (gavage;corn oil)

100 300 Decreases in body weight and diarrhoea inhigh-dose dams. No remarkable foetaleffects.

Rabbit Oral (gavage) 50 150 Decreases in body weight and foodconsumption in high-dose dams.No remarkable foetal effects.

Pre- and Post-Natal Effects

Rat Diet 652 Slight decreases in foetal body weights andin mean number of live pups at the highestdose.

1 In all cases, the NOEL values were taken to be the NOAEL values for the study as only NOEL values were

determined.2 Male and female doses combined

3.3.9. Toxicokinetics

More than 30 non-clinical pharmacokinetics and/or toxicokinetics studies investigatingabsorption, distribution, metabolism, and excretion of triclosan have been reviewed for thisdossier. The kinetics of triclosan and its metabolites have been studied in mice, rats,hamsters, guinea pigs, rabbits, dogs, and monkeys. In these studies, the oral, intravenous,dermal, intraperitoneal, intravaginal, and intraduodenal routes of administration have beenexamined. In mice, rats, and hamsters, single, stand-alone studies were conducted thatinvestigated all pharmacokinetic (PK) parameters (i.e., absorption, distribution, metabolism,

excretion, ADME).

Unlabelled triclosan, 14C-labelled triclosan, and 3H-labelled triclosan have been used to studythe pharmacokinetics of triclosan. The pharmacokinetic data assessed included themaximum concentrations in blood and tissues (Cmax), the corresponding time required toreach the peak concentrations (Tmax), the area under the curve (AUC), the elimination half-life (t1/2), and the pattern of excretion. In general, the pharmacokinetic studies used similarmethodologies to monitor and quantitate the pharmacokinetic data. Following a single orseries of extraction and/or partitioning step(s), the blood and tissue samples were analysedby thin layer chromatography (TLC), gas chromatography (GC) with electron capture, highperformance liquid chromatography (HPLC), and liquid scintillation counting. Whole bodyautoradiography was used to determine the relative location of the 14C-labelled triclosan in

distribution studies.



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Three stand-alone PK/ADME studies for triclosan were conducted in mice, rats, andhamsters [van Dijk, 1994 (80); van Dijk, 1995 (81); van Dijk, 1996 (82)]. These studieswere similar in design, with the exception that the rat study did not investigate themetabolism of triclosan. The remainder of the data available were generated from studieswhere toxicokinetics was not the primary focus. These kinetic data were derived fromtoxicokinetic studies conducted in conjunction with toxicology studies [Caudal et al ., 1975

(83); Parkes, 1986 (84); Hohensee and Berke, 1991 (85)]. Overall, however, the threePK/ADME studies in mice, rats, and hamsters provide definitive single-dose data, withsupporting evidence from the remaining studies.The pharmacokinetic data for triclosan in all species investigated, including the route ofadministration and the pharmacokinetic parameters assessed, are summarised in Table 22.

Table 22: Kinetic Parameters Measured in Non-clinical Studies

Species Route1 Parameter Measured Reference

Mouse oral Cmax, Tmax, T1/2, AUC van Dijk, 1995 (81)

Rat oral Cmax, Tmax, T1/2, AUC Lin and Smith, 1990 (86), Black et al .,1975 (27),

van Dijk, 1996 (82)

Rat i.v. T1/2 Siddiqui and Buttar, 1979 (87)

Rat i.v.g. Cmax, Tmax Siddiqui and Buttar, 1979 (87)

Guinea Pig oral T1/2 Black et al ., 1975 (27)

Hamster oral Cmax, Tmax, T1/2, AUC van Dijk, 1994 (80)

Rabbit oral Cmax, Tmax Hong et al ., 1976 (24)

Dog oral Cmax, Tmax Ciba-Geigy, 1976a (88)

Dog i.v. Cmax, Tmax Stierlin, 1972a (89)

Monkey oral Cmax, Tmax Parkes, 1978b (90), Ciba-Geigy, 1976a(88), Ciba-Geigy, 1977a (91)

Monkey dermal Cmax, Tmax Hazleton Laboratories, 1979b (30)1 i.v.=intravenous; i.v.g.=intravaginal

3.3.9.1.1 Single Dose Data

PK/ADME parameters have been examined following single oral doses of 14C-labelledtriclosan to mice, rats, and hamsters [van Dijk, 1994 (80); van Dijk, 1995 (81); van Dijk,1996 (82)]. The single-dose data are summarized in Table 23. Other details from thesestudies are included under the appropriate heading (i.e., absorption, distribution,metabolism, excretion).

Table 23: Summary of Plasma Toxicokinetics of 14C-Triclosan after Single Oral

Administration at Two Dose Levels to Rodents

Plasma Levels1

Species Route Target Dose

(mg/kg bw)

Sex Cmax

(µg/g)

Tmax (hr) t1/2 (hr) AUC2

(mg hr/L)

C96

(mg/L)

2 M 19.48 4 9.1 166.1 0.02

200 M 212.8 4 11.8 4,505 1.1716

8.79 12 F

7.67 4

8.9 119.3 0.007

267.2 2

Mouse3 oral

200 F

263.3 4

9.9 6,322 0.742

Rat3 oral 2 M 4.772 1 12.6 63.9 0.086



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Plasma Levels1

Species Route Target Dose(mg/kg bw)

Sex Cmax

(µg/g)Tmax (hr) t1/2 (hr) AUC2

(mg hr/L)C96

(mg/L)

4.458 4

153.4 1200 M

183.3 4

10.0 3,237 1.25

2 M 7.684 1 29.1 178.0 0.181

200 M 359.171 1 32 6,010 7.784

2 F 7.357 1 24.5 79.8 0.071

Hamster3 oral

200 F 384.920 1 27.0 4,298 4.5561 Cmax =maximal concentrations in blood and tissues, Tmax =the time required to reach the peak concentration,AUC= the plasma area under the concentration-time curve, t1/2= the elimination half-life of the radioactivity,C96=plasma concentration at 96 hr2 AUC values representative of time of animal sacrifice: 96 h (mice, rats); 168 h (hamsters) (Tmax)3 Data from: van Dijk, 1994 (80); van Dijk, 1995 (81); van Dijk, 1996 (82)4 Animals sacrificed at 96 h (mice, rats) and 168 h (hamsters).

Triclosan is rapidly absorbed as indicated by the time to reach Cmax. Two peaks (2 x Tmax) inplasma triclosan levels were detected in mice and rats, with peak plasma concentrationsoccurring after 1 and 4 hours in these 2 species. Cmax values obtained following 2 versus 200 mg/kg body weight/day did not reflect the 100-fold increase in dose of triclosan. Acomparison of the Cmax data for the low and high doses indicates that the process ofabsorption may be at least partially saturated at the high dose level and elimination may beenhanced. Triclosan appeared to have high systemic exposure following oral administration,based on urinary excretion data (i.e., high levels of radioactivity were excreted in the urinefollowing dosing, indicating good absorption).

Single Dermal Dose

With respect to dermal applications of triclosan, a single-application study in newbornRhesus monkeys using a triclosan-containing soap solution (1 mg/mL, 0.1%) resulted in aTmax of 12 hours and a Cmax of 0.5 to 0.7 ppm (500 to 700 ng/mL) [Parkes, 1978a (29)]. Incomparison to the single-exposure monkey data, rats displayed 2 peaks in plasmaconcentration 2 hours (0.278 ppm or 278 ng/mL) and 6 hours (0.264 ppm or 264 ng/mL)following the dermal application of triclosan in an ethanol solution to a 7.5 cm2 section of ratskin [Black and Howes, 1975 (23)].

3.3.9.1.2 Repeated Dose Data

Repeated Dose Data from Pharmacokinetic/ADME Studies

In the PK/ADME studies in mice, rats, and hamsters considered to be definitive, plasmaconcentrations and, in the case of rats, AUC, were determined following 13 days of triclosanadministration in the diet. PK analyses were conducted on blood and tissue samples takenfollowing a dose of 14C-labelled triclosan on Day 14 of repeated dosing. Plasmaconcentrations at Cmax were comparable following the bolus radiolabelled dose on Day 14compared to the single dose in both rats and hamsters. In contrast, the Cmax level for asingle oral dose of triclosan in plasma following repeated daily exposure was decreased inmice, compared to plasma levels following a single dose. These findings are summarized inTable 24.

Table 24: Summary of Cmax Values Following Single and Repeated Doses of 2 mg/kgbw/day of Triclosan1



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Species Time (h) Single Dose Cmax (µg/peq/g)2

Repeated Dose Cmax (µg/peq/g)1

Mouse 4 14.326 4.977

Rat 1-2 4.772 4.491

Hamster 1-2 4.883 5.003

1 Data from: van Dijk, 1994 (80); van Dijk, 1995 (81); van Dijk, 1996 (82)2 µg parent equivalents per gram plasma

Data from the study in rats that examined AUC following both single and repeated doses oftriclosan indicate AUC levels in plasma did not change after repeated dosing for 14 days, asshown in the table below.

Table 25: Comparison of Calculated AUC Values Following Single and Repeated Doses ofTriclosan in Rats

Dose(mg/kg bw/day)

Single Dose AUC(ng*hr/mL)

Repeated Dose AUC(ng*hr/mL)

2 64,000 77,400200 3,237,000 3,581,000

Repeated Dose Toxicokinetic Data from Oral Toxicology Studies

Plasma levels of triclosan were determined following 12 to 14 days of oral (dietary)administration to mice of daily doses of 10 mg/kg body weight were 22,500, 22,000, and23,600 ng/mL on Days 12, 13, and 14 of dosing, respectively [Huntingdon Life Sciences,1997 (92)]. The AUC value calculated for these data was 489,000 ng*hr/mL.

Plasma levels of triclosan were also determined in the chronic carcinogenicity bioassays

conducted in mice, rats, and hamsters [Pharmaco LSR, 1995 (66); Ciba-Geigy, 1986 (67);Huntingdon Life Sciences, 1999 (68)]. Tables 26 through 28 inclusive present the bloodlevels of triclosan from these 3 carcinogenicity studies. For purposes of comparison, Table29 presents triclosan levels following at least 6 months of daily oral (dietary) doses in thesestudies, including dose-normalized data. These data indicate that in chronic dosing studies,plasma levels in mice were slightly higher or comparable to plasma levels in rats (based ondose-normalized data) and much higher than plasma levels in hamsters (greater than 4- to5-fold).

Table 26: Plasma Concentrations (ng/mL) of Triclosan in Mice Following Chronic DietaryAdministration

Interim(6 months)

Termination(18 months)

Males Females Males Females

Dose(mg/kg/d)

Mean SD Mean SD Mean SD Mean SD

10 16,800 4,260 21,900 8,220 20,600 11,100 21,100 7,300

30 58,900 15,300 75,700 11,500 62,400 26,100 98,900 21,300

100 134,600 25,000 172,100 27,200 150,200 37,400 169,000 69,400

200 177,600 49,300 191,500 37,700 197,200 43,100 236,500 59,900

Source: Pharmaco LSR, 1994 (66).

Table 27: Plasma Concentrations (ng/mL) of Triclosan (FAT 80’023/S) in Rats FollowingChronic Dietary Administration1



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Males Females

Interim

(52 weeks)

Termination

(104 weeks)

Interim

(52 weeks)

Termination

(104 weeks)

Dose

(mg/kg/d)2

Mean SD Mean SD

Dose

(mg/kg/d)3

Mean SD Mean SD

12-9 21,808 9,008 10,576 3,392 17-11 28,160 12,928 26,496 18,03

40-34 55,024 12,656 43,296 15,760 56-34 70,624 22,736 42,048 31,88

127-107 120,368 44,464 86,784 26,416 190-114 170,656 32,928 138,560 43,64

Source: Ciba-Geigy, 1986 (67).1 Note that the original total values for triclosan (free + conjugates) were in units of ng/mL in blood, not plasma.Conversions to values in ng/mL plasma (presented in this table) were based on an average blood volume of 64mL/kg (range: 58-70 mL/kg) and an average plasma volume of 40 mL/kg (range: 36-45 mL/kg). Source for valuefor rat plasma volume: McGuill and Rowan, 1989.2 Average daily dose calculated at approximately 52 weeks (first number) and 104 weeks (second number) formales consuming dietary doses of 300, 1,000, and 3,000 ppm. Daily doses (mg/kg/d) were decreased by15-20%at 104 weeks compared to 52 weeks.3 Average daily dose calculated at approximately 52 weeks (first number) and 104 weeks (second number) formales consuming dietary doses of 300, 1000, and 3,000 ppm. Daily doses (mg/kg/d) were decreased by 35-40%

at 104 weeks compared to 52 weeks.

Table 28: Plasma Concentrations (ng/mL) of Triclosan (FAT 80’023/S) in HamstersFollowing Chronic Dietary Administration

Interim

(52 weeks)

Termination

(95 weeks for Males; 90 weeks for Females)

Males Females Males Females

Dose

(mg/kg/d)

Mean SD Mean SD Mean SD Mean SD

12.5 1,411 467 578 188 1,322 232 655 148

75 5,310 972 2,578 847 8,162 3,404 3,683 664

250 19,390 3,465 9,942 3,055 50,985 34,197 43,624 56,279

Source: Chasseaud et al., 1994 (93), located in Huntingdon Life Sciences, 1999 (68)

Table 29: Steady State Concentrations of Triclosan in Mice, Rats, and Hamsters FollowingChronic Dosing

Interim1 (ng/mL) Termination2 (ng/mL)Species Dose (mg/kg

bw/d)

Sex

Mean SD Mean SD

M 134,600 25,000 150,200 37,400100

F 172,100 27,200 169,000 69,400

M 177,600 49,300 197,200 43,100200

F 191,500 3,700 236,500 59,900

M 888-1,346 -- 986-1,502 --

Mouse3

Dose-Normalized

F 958-1,721 -- 1,183-1,972

--

127-1075 M 120,368 44,464 86,784 26,416

190-1146 F 170,656 32,928 138,560 43,648

M 947 -- 811 --

Rat4

Dose-Normalized

F 898 -- 1,216 --

M 19,390 3,565 50,985 34,197250

F 9,942 3,055 43,624 56,279

Hamster3

Dose-Normalized M 78 -- 204 --



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F 40 -- 174 --1 Mice=6 months, Rats=52 weeks, Hamsters=52 weeks2 Mice=18 months, Rats=104 weeks, Hamsters=95 weeks (M); 90 weeks (F)Abbreviations: M=Male, F=Female3 Plasma concentration4 Conversions from blood concentration to plasma concentration were based on an average blood volume of 64mL/kg (range: 58-70 mL/kg) and an average plasma volume of 40 mL/kg (range: 36-45 mL/kg). Source for ratplasma volume: McGuill and Rowan, 1989.5 Average daily dose calculated at approximately 52 weeks (first number) and 104 weeks (second number) formales consuming dietary doses of 3,000 ppm. Daily doses (mg/kg/d) were decreased by 15-20% at 104 weekscompared to 52 weeks6 Average daily dose calculated at approximately 52 weeks (first number) and 104 weeks (second number) formales consuming dietary doses of 3,000 ppm. Daily doses (mg/kg/d) were decreased by 35-40% at 104 weekscompared to 52 weeks.

In summary, in the 3 rodent studies presented in Tables 23 and 24, target dose levels of 2and 200 mg/kg body weight were used in each study, and thus relevant comparisons ofexposure between species can be made following single doses of triclosan. The AUC data(measured over 96 hours in mice and rats and 168 hours in hamsters) show that the mouseand hamster have much higher levels of exposure (almost 3-fold) compared to the rat. The

levels of exposure in the mouse and hamster are shown to be similar. Plasma levels at 96hours were much higher in hamsters than in mice and rats. Based on data from the ratPK/ADME study, there was no change in AUC after 14 days of repeated daily dosing. Datafrom chronic dosing studies showed that plasma levels in mice were slightly higher orcomparable to plasma levels in rats (based on dose-normalized data) and much higher thanplasma levels in hamsters (greater than 4- to 5-fold). Plasma levels in the chronic mousestudy were comparable to those in the 14-day study (16,800 and 21,900 ng/mL in malesand females, respectively).

Repeated Dermal Doses

In the 90-day study in newborn Rhesus monkeys that used a 0.1% triclosan soap solution,

plasma levels ranged from 0.17 to 0.97 ppm (170 to 970 ng/mL) [Hazleton Laboratories,1979b (30)]. The data from this study showed that plateau levels of triclosan in plasmawere reached within 15 days of daily treatment. Other pharmacokinetic data (e.g., AUCs ort1/2) were not available for triclosan administered via the dermal route.

3.3.9.1.3 Bioaccumulation/Bioretention

The accumulation or retention of triclosan in organs and tissues was investigated in thedefinitive PK/ADME studies in rodents [van Dijk, 1994 (80); van Dijk, 1995 (81); van Dijk,1996 (82)]. Tissue distribution data from the hamster study showed persistently higherplasma levels of triclosan compared to levels in liver, kidney, and lung followingadministration of either the low or high dose of 14C-labelled triclosan (2 or 200 mg/kg body

weight/day) [van Dijk, 1994 (80)]. Liver, kidney, and lung contained the highest levels oftriclosan following oral administration, followed by the gastrointestinal tract. Levels oftriclosan in plasma, liver, kidney, and lung following single versus repeated oral doses of14C-labelled triclosan are shown in Table 30. A comparison of levels following single doseversus repeated shows no evidence of accumulation or retention of triclosan in liver, kidney,or lung. Taken altogether, the data indicate that triclosan is efficiently eliminated fromorgans/tissues of the hamster and that there is no accumulation in these tissues afterrepeated exposure to triclosan.

Table 30: Levels of Triclosan in Plasma, Liver, Kidney, and Lung Following Single orRepeated Doses of 14C-Labelled Triclosan in Hamsters1

2 mg/kg bw/day 200 mg/kg bw/dayOrgan/TissueMale Female Male Female



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SingleDose

RepeatedDose

SingleDose

RepeatedDose

SingleDose

RepeatedDose

SingleDose

RepeatedDose

Plasma 0.317 0.209 0.093 0.089 16.175 7.898 7.139 5.206

Liver 0.068 0.037 0.017 0.015 3.433 1.831 1.361 1.212

Kidney 0.138 0.061 0.019 0.016 3.254 1.488 1.249 0.938

Lung 0.091 0.062 0.027 0.028 4.415 2.204 1.915 1.4701 Levels in µg of parental equivalents/gram of tissue.

Additional evidence of a lack of bioaccumulation or bioretention of triclosan is provided bythe plasma AUC data generated in the definitive rat PK/ADME study [van Dijk, 1996 (82)].In this study, the AUC value calculated following an oral dose of 14C-labelled triclosan onDay 14 of a repeated triclosan exposure (diet) regimen was comparable to the AUC valuecalculated following a single oral dose (see Table 25). The similarity of the single dose andrepeated dose AUC values suggests that the amount of triclosan absorbed is equal to thateliminated. Together with the similarity in plasma Cmax values following repeated or singledoses (shown in Table 24), the data show that accumulation of triclosan is unlikely to occurwith repeated exposure.

It should be noted that tissue distribution data in the murine PK/ADME study showed that,at the oral dose of 200 mg/kg body weight/day for 14 days, levels of triclosan in the liver ofmale mice were approximately 2 times higher than levels in plasma [van Dijk, 1995 (81)].

While definitive studies examining tissue distribution over time have not been conducted fordermal application studies, measurements of plasma levels of triclosan in the 90-day studyof newborn Rhesus monkeys washed with a soap solution containing 0.1% triclosan showedlevels of 170 to 970 ng/mL triclosan in plasma. A comparison with Cmax plasma levels of500 to 700 ng/mL at the Tmax of 12 hours after a single application suggests that there is noevidence of bioaccumulation/bioretention of triclosan after repeated dermal exposures.

In summary, both tissue distribution and plasma AUC data in hamsters and rats,respectively, provide evidence of a lack of bioaccumulation/bioretention of triclosan. Tissuedistribution data in the mouse show that increased levels of triclosan are found in mouseliver compared to plasma.

3.3.9.2 Absorption

3.3.9.2.1 Oral Absorption

Overall, the data from studies in rodents and non-rodents suggest that triclosan is rapidlyand highly absorbed from the gastrointestinal tract by all species, with maximum plasmaconcentrations typically being reached between 4 to 8 hours [van Dijk, 1995 (81); Lin and

Smith, 1990 (86); Black et al ., 1975 (27); Siddiqui and Buttar, 1979 (87); van Dijk, 1994(80); Ciba-Geigy, 1976a (88); Stierlin, 1972a (89); Parkes, 1978b (90)]. A directestimation of absorption was calculated from the comparison of AUC values between theoral and i.v. routes of administration in the rat. In this study, it was estimated that an oraldose was 70 to 80% absorbed in rats, based on i.v. and oral AUC values calculated from a 5mg/kg body weight dose [Stierlin, 1972a (89)]. Likewise, in 1 dog study a comparison ofurinary and faecal recovery following both oral and i.v. administration suggests anabsorption rate of 50% in this species.

Autoradiography studies in mice, rats, and hamsters, also observed high levels ofradioactivity in well-perfused organs within 30 minutes of an oral administration of triclosan[Kanetoshi et al ., 1988 (94); Howes et al ., 1989a (95); Howes et al ., 1989b (96); Lin andSmith, 1990 (86); Stierlin, 1972a (89); Howes and Moule, 1989 (97)]. Altogether, thesefindings suggest that triclosan is highly absorbed following oral administration, with nospecies-related differences.



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3.3.9.3 Distribution

A number of studies have been carried out to determine the distribution of 14C-labelledtriclosan in the tissues of mice, rats, hamsters, and monkeys.

In tissue distribution analyses conducted in mice following single and repeated doses of 2 or200 mg/kg body weight/day of triclosan, levels of triclosan were comparable between liverand plasma [van Dijk, 1995 (81)]. However, with repeated dosing, radioactivity levels inthe liver remained consistently higher than levels in plasma. In other studies in miceadministered radiolabelled triclosan via the oral route, the sites with the highest levels ofradioactivity were typically the stomach, GI tract, and gall bladder [Kanetoshi et al ., 1988(94); Howes et al ., 1989a (96); Howes et al ., 1989b (97)]. Organs/tissues that are well-perfused (e.g., heart, lung, liver, kidney) also routinely displayed detectable levels oftriclosan. High levels of radioactivity were still detected in the small intestine and gallbladder (the highest of all mouse tissues with levels of 1,130 µg Irgasan DP300equivalents/g tissue) at 24 hours after a single oral dose of triclosan in ddY mice (100 µCi)[Kanetoshi et al ., 1988 (94)]. Similar findings were observed following i.v. administration

of triclosan in mice, with the highest levels of radioactive triclosan detected in the tissues ofthe liver, gut, and gall bladder [Stierlin, 1972a (89); Ciba-Geigy, 1977a (91)].

In rats administered triclosan via the oral route, the tissues with the highest levels ofradioactivity included the pituitary gland, bladder, large intestine, kidneys, liver, stomach,and gingiva [Lin and Smith, 1990 (86); Stierlin, 1972a (89)]; however, after 24 hoursfollowing i.v. administration of triclosan, the only tissues with levels >1.3 µg/g were foundto be liver, lung, and kidney [Stierlin, 1972a (89)]. In an examination of plasma, liver, andkidney levels of triclosan following oral doses of 2 or 200 mg/kg body weight/day, plasmaand liver concentrations were comparable and kidney levels lower than plasma, even afterrepeated high doses of triclosan [van Dijk, 1996 (82)].

In the definitive PK/ADME study in hamsters, tissue distribution analyses showed thatfollowing either single or repeated doses of 2 or 200 mg/kg body weight/day of 14C-labelledtriclosan, the highest levels of triclosan were: plasma >> kidney ~liver ~ lung (see Table30) [van Dijk, 1994 (80)]. Levels in the excretory organs kidney and liver as well as inlungs were about 3 to 6 times lower compared to plasma levels. In another study in thehamster, the highest levels of radiolabelled triclosan were in the gall bladder, stomach, andGI tract, with lower levels in the kidney and liver (no levels given) [Howes and Moule, 1989(97)].

In monkeys washed daily with 0.1% triclosan for 90 days, triclosan species were detected inthe lung (0.2 to 1.3 ppm), liver (0.1 to 0.5 ppm), kidney (0.1 to 0.9), and skin (0.6 to1.4 ppm), confirming that triclosan distributes to the liver, lung, and kidney in primates

[Hazleton Laboratories, 1979b (30)].

Overall, these data suggest that in rodent species the GI tract, kidney, liver, and gallbladder are the target organs for the disposition of triclosan. A significant contribution tothis observation was postulated to result from biliary excretion leading to enterohepaticcirculation. In the mouse, levels of triclosan in liver were higher than plasma following long-term repeated dosing.

3.3.9.3.1 Enterohepatic Circulation

Based on whole-body autoradiography studies that identified high levels of radioactivity inthe gall bladder and GI tract, and data showing the presence of 2 peak concentrations in theplasma following single or repeated dosing, it has been suggested that mice and rats exhibitenterohepatic circulation [van Dijk, 1995 (81); van Dijk, 1996 (82); Howes et al ., 1989a(94); Howes et al ., 1989b (96); Kanetoshi et al ., 1988 (94); Ciba-Geigy, 1977a (91);



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Stierlin, 1972a (89)]. As such, these species would experience an enhanced local exposureto triclosan in the liver and GI tract. An i.v. study was conducted to investigate thepotential for the enterohepatic circulation of triclosan in rats with biliary-fistula, [Ciba-Geigy,1975b (98)]. Following a 5 mg/kg body weight i.v. dose, 73% of the dose was detected inthe bile and then readministered into the duodenum. Of this re-administered dose, 38.8%(28.4% of the original dose) was found in the bile. Thus, these findings provide evidence

that triclosan was reabsorbed from the GI tract. Although not definitive, additional evidencethat supports the enterohepatic circulation in mice and rats comes from excretion data.Specifically, data from a number of studies have shown that triclosan is excreted primarilyvia the faecal route in mice and rats, regardless of the route of administration ( i.e., evenfollowing an i.v. dose) [Hazleton Laboratories, 1979b (30); Lin and Smith, 1990 (86);Howes et al ., 1989b (96); van Dijk, 1995 (81); van Dijk, 1996 (82)]. These data suggest astrong GI component to the distribution of triclosan in these species, even following anintravenous dose. In contrast, although high levels of triclosan were detected in thegallbladder and GI tract of hamsters, there were no pharmacokinetic or distribution datacomparable to those in mice and rats that would suggest any extensive enterohepaticcirculation in this species [Howes and Moule, 1989 (97)].

3.3.9.3.2 Plasma Protein Binding

The plasma protein binding of triclosan in human, mouse, and hamster blood wasinvestigated in an in vitro study [Sagelsdorff and Buser, 1995 (99)]. Plasma was incubatedwith Irgasan DP300 at final concentrations of 3.2, 6.4, and 16 µg/mL. An equilibriumdialysis method was used and the ratio of the bound to the unbound fractions was constantover the tested concentrations (i.e., no saturation of binding was observed during testing).The plasma protein binding was determined to be 98.4 to 99.2, 98.1 to 98.7, and 98.7 to99% in humans, mice, and hamsters, respectively [Sagelsdorff and Buser, 1995 (99)].Thus, triclosan is highly bound in the plasma, with no species differences observed.

3.3.9.4 Metabolism

The metabolism of triclosan has been studied in a number of species including mice, rats,hamsters, dogs, and monkeys. An early study in the rat showed that metabolites oftriclosan occurred mainly via aromatic hydroxylation of the ortho and meta positions to theether bond of the benzene rings and to a smaller degree by scission of the ether bond.Scission of the ether bond produces 2,4-dichlorophenol (found in faeces and urine) and4-chlorocatechol (excreted in the urine) [Tulp et al ., 1979 (100)]. Two key studies haveexamined the metabolic pathway of triclosan in mice [van Dijk, 1995 (81)] and hamsters[van Dijk, 1994 (80)]. In the liver and skin, triclosan is primarily metabolised toglucuronide and sulfate conjugates, as well as other non-parent metabolites.

3.3.9.4.1 Biotransformation of triclosan

Triclosan is metabolised to parent sulfate and parent glucuronide conjugate compounds inall species examined to date. However, the relative ratio of these conjugates differs amongspecies. The specific identity of the sulfate and glucuronide metabolites has beendetermined in mice, rats, and hamsters using TLC and gas-chromatography massspectrometry (GC-MS) [van Dijk, 1995 (81); van Dijk, 1996 (82); van Dijk, 1994 (80)]. Inaddition to the parent compound and parent conjugates (glucuronide and sulfate), severalnon-parent metabolites and conjugates were detected [parent (M1), parent glucuronide(M7), parent sulfate (M4), non-parent metabolites (M2 and M3) and correspondingconjugates]. The non-parent metabolites are products of phase 1 metabolism, which aresubsequently conjugated (phase 2 metabolites).

Following repeated dosing at target dose levels of 200 mg/kg body weight/day, there weremore than 7-fold and 2-fold decreases in the levels of the non-parent metabolites in theurine of male mice (52.5 to 7.3%) [van Dijk, 1995 (81)] and hamsters (38.5 to 16.4% )



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[van Dijk, 1994 (80)], respectively. Concomitant increases in the appearance ofglucuronide conjugates in the urine were observed for both species, suggesting a decreaseof phase 1 metabolism with repeated doses of triclosan.

As well as identifying and characterizing the triclosan metabolites in mice [van Dijk, 1995(81)] and hamsters [van Dijk, 1994 (80)], much effort has been made in stand-alone or

specific portions of studies to identify the predominant triclosan species in the urine, faeces,and plasma of mice [van Dijk, 1995 (81)], hamsters [van Dijk, 1994 (80)], and dogs[Hohensee and Berke, 1991 (85)]. These data are summarized in Table 31 below. Ingeneral, the sulfate, glucuronide, and free species are predominantly found in the plasma,urine, and faeces, respectively.

Table 31: Predominant Triclosan Species in Plasma, Urine, and Faeces of Animal SpeciesFollowing Single and Repeated Dosing

Species Route Duration Plasma Urine Faeces Reference

single Free: 64(M)Sulfate: 73(F)

Low dose:Glucuronide

23% (M)2;High dose: Free65% (M);Glucuronide70% (F)

Free66-96

oral

repeated Sulfate78-90

Low dose:Free, 18-38%;High dose:Glucuronide,63-75%

Free68-96

single - Free3,25-33%

Free74-86

Mouse (% ofradio-activity

recovered)

i.v.

repeated - Free 43% (M);Glucuronide

32% (F)

Free68-75

van Dijk,1995 (81)

Rat (ng/mL) oral single/ repeated Sulfate30 d=11,880 (M)92 d=13,300 (M)

Free2,308 (M)57,350 (F)

Free17,000ng/g (M)150,000ng/g (F)

van Dijk,1996 (82)

single Glucuronide28-36

Glucuronide1

56-77Free55-87

oral

repeated Glucuronide24-56

Glucuronide60-87

Free57-91

single - Glucuronide27-58

Free20-51

Hamster(%)

i.v.

repeated - Glucuronide33-56 Free27-65

van Dijk,1994 (80)

Dog (ng/mL) oral 30-day Sulfate9,688

Glucuronide2,541

Free46,367

Hohenseeand Berke,1991 (85)

Monkey(ppm, at 24hr)

oral single dose Sulfate1 1.61-3.18

Glucuronide21.3-78.8

Free114-294

Parkes,1978b (90)

Abbreviations: M, male; F, female1 Single, low-dose female had 43.3% free and 12.5% glucuronide2 Single, low-dose male had no detectable glucuronide3 Single, low-dose female had 39% glucuronide, male: 0% glucuronide.

A preliminary study was conducted to investigate the metabolism of triclosan in monkeysand dogs administered single oral doses of radiolabelled triclosan (5 mg/kg) [Ciba-Geigy,1976a (88)]. Blood levels of radiolabelled triclosan compounds in dogs and monkeys were



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6.08 µg/mL (T=3 hours) and 1.24 µg/mL (T=8 hours), respectively. The nature of themetabolites was revealed following incubation of samples with specific glucuronidase andarylsulfatase enzymes (i.e., glucuronide and sulfate conjugates) and subsequent analysis byTLC. Prior to enzyme hydrolysis the levels of parent compound were less than 0.8%;however, following enzyme hydrolysis the levels of parent triclosan compound were greaterthan 80%.

Data on the systemic (not dermal) metabolism of dermally-applied triclosan are availablefrom a 90-day bathing study conducted in Rhesus monkeys and its accompanying pilotstudy [Parkes, 1978a (29), Hazleton Laboratories, 1979b (30)]. In the pilot, single-dosestudy, 2 Rhesus monkeys (3 days old) were washed with a soap solution containingtriclosan (1 mg/mL, volume was not disclosed) [Parkes, 1978a (29)]. Blood samples takenat 1, 3, 5, 8, 12, and 24 hours after washing showed that both glucuronide and sulfateconjugates were present and that no free, unconjugated triclosan was detectable. Bloodlevels reached plateau levels (0.43 to 0.68 ppm) by 8 to 12 hours after washing and weremaintained for 8 to 24 hours. In the 90-day study, blood levels of total triclosan reached aplateau by 15 days and ranged from 0.17 to 0.97 ppm [Parkes, 1978a (29)]. Triclosan waspresent almost exclusively as glucuronide and sulfate conjugates in the blood; however, the

glucuronide conjugate was predominant in samples from Days 1 to 2, after which point thesulfate conjugate predominated, such that sulfate conjugate levels in blood samples at theend of the study were 80 to 90% of the dose. Urinary concentrations ranged from 0.3 to4.8 ppm and primarily contained the glucuronide conjugate, while faecal levels ranged fromless than 0.1 to 10.5 ppm (primarily free triclosan). Overall, the monkey data show thattriclosan is absorbed and metabolised to both glucuronide and sulfate conjugates followingdermal application and that repeated dermal exposures to triclosan result in the formationof the sulfate conjugate of triclosan as the primary metabolite.

3.3.9.4.2 Dermal Metabolism

An in vitro diffusion skin cell model was used to assess the ability of the skin (rat and

human) to metabolise triclosan applied using an ethanol:water (9:1) vehicle [Moss et al .,2000 (21)]. Glucuronidation and sulfation of triclosan were demonstrated to occur in thismodel (i.e., the conjugates were found in the collecting reservoir following absorptionthrough the skin), with the glucuronide being the primary metabolite at levels up to 1.4%.These findings were supported by in vivo studies with rats in which 0.4 and 1.5% of theparent glucuronide were reported to occur in the urine and skin, respectively, following thedermal application of triclosan [Moss et al ., 2000 (21)]. These data show that triclosan ismetabolised in the skin.

3.3.9.5 Excretion

The excretion of 14C-labelled and 3H-labelled triclosan was studied in mice, rats, guinea pigs,

hamsters, dogs, and monkeys. These studies were the most frequently conducted of alltriclosan pharmacokinetic studies. In particular, studies to ascertain the elimination half-life, the routes of excretion, and enterohepatic circulation were conducted for triclosan.

3.3.9.5.1 Elimination Half-Life

The terminal plasma half-life of orally administered 14C-labelled triclosan and its metaboliteswas comparable in rats and mice, but much greater in hamsters (up to 3-fold). In mice,rats, and hamsters the half-life of radiolabelled triclosan is 9 to 12, 10 to 13, and 24 to 32hours, respectively (Table 23) [van Dijk, 1995 (81); Lin and Smith, 1990 (86); van Dijk,1996 (82); van Dijk, 1994 (80)]. In hamsters, the much longer half-life of radioactivetriclosan is likely attributed to the long residence time of the non-parent M6 and M8/9metabolites. Different routes of administration [e.g., intraperitoneal (i.p.)] also have beenassociated with extended or longer half-lives. In rats given tritiated DP300 by i.p. injection,the half-life of radioactive triclosan was 18 hours as compared to 9 to 12 hours [van Dijk,1996 (82)].



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3.3.9.5.2 Routes of Excretion Following Oral Applications of Triclosan

In mice, rats, hamsters, and monkeys, unlabeled and radiolabelled 14C-triclosan was used toassess the routes of excretion for triclosan following oral doses of triclosan. For studiesusing radioactive triclosan, the predominant route of excretion in mice and rats was the

faeces, whereas in hamsters it was the urine. Based on a survey of the available data, theexcretion rates for these species are presented in Table 32.

Table 32: Routes of Excretion of Triclosan Following Oral Dosing in Rodent Species

Faeces (%) Urine (%) Reference

Mouse 25-89 26-44 Howes et al ., 1989a (95); Howes et

al ., 1989b (96); van Dijk, 1995 (81)

Rat 81-84 4-12 van Dijk, 1996 (82); Lin and Smith,1990 (86); Hong et al ., 1976 (24)

Hamster 0.1-0.3 60-80 Howes and Moule, 1989 (97); vanDijk, 1994 (80)

Routes of excretion in monkeys, have been shown to be urinary in a study using unlabelledtriclosan administered to monkeys via oral gavage [Parkes, 1978b (90)], and faecal in astudy using dermal applications (washing with soap containing 0.1% triclosan) [HazletonLaboratories, 1979b (30)].

3.3.9.5.3 Routes of Excretion Following Dermal Applications of Triclosan

Excretion following dermal applications of triclosan showed that the faecal routepredominated in rats, where triclosan was 70 to 90% excreted in the faeces compared to 3to 4% eliminated in the urine [Ciba-Geigy, 1976b (25)]. Comparable data showingprimarily faecal excretion of triclosan regardless of the formulation of the dose were found

in other rat studies [Hong et al., 1976 (24); Moss et al., 2000 (21)].

In contrast to rats, rabbits exposed to triclosan in a dermally-applied solution or creamshowed moderately greater urinary excretion compared to faecal elimination (47 to 53% vs. 38% of the applied triclosan solution excreted in the faeces; 29 to 48% in urine vs. lessthan 2% in faeces following the cream application) [Ciba-Geigy, 1976b (25)].

Triclosan levels in the urine and faeces of monkeys bathed daily from birth to 90 days with15 mL of a soap solution containing triclosan (1 mg/mL) were found to range from 0.3 to4.8 ppm in the urine and 0.1 to 10.5 ppm in the faeces [Hazleton Laboratories, 1979b(30)]. These data suggest that the faecal route may have dominated in the excretion oftriclosan. For the purposes of comparison, it should be noted that the primary route of

excretion in humans exposed to triclosan via the dermal route is urinary.

In summary, the primary route of excretion following dermal exposure to triclosan differsbetween species, with the faecal route predominating in the rat, but the urinary routepredominating in the rabbit. Neither faecal nor urinary elimination appear to be stronglyfavoured in the case of primates based on the available data.



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3.3.9.6 Summary of Pharmacokinetic and Toxicokinetic Data in Animals

Triclosan is rapidly absorbed (via the oral, i.p., and i.v.g. routes of administration) in allspecies. Specific studies conducted in rats indicated that the level of absorption wasbetween 70 to 80% following oral administration in this species. Subsequent to absorptionafter oral administration, 2 peaks in plasma triclosan levels were detected in mice and ratsat 1 and 4 hours, which is indicative of enterohepatic circulation. Evidence from bothhamster and rat studies provide evidence of a lack of bioaccumulation/bioretention oftriclosan following repeated oral doses.

Triclosan is widely distributed in organs and tissues, with well-perfused, and excretory,organs such as liver and kidney, as well as lung, heart, GI tract, and gall bladder showinghighest levels following oral, dermal, or i.v. administration in rodents and monkeys. Levelsof triclosan in mouse liver were higher than in plasma following repeated dosing withtriclosan.

Following absorption, the parent triclosan compound was found to be metabolised to both

glucuronide and sulfate conjugates. Although different ratios of the individual glucuronideand sulfate conjugates were observed among species, no unique species-specificmetabolites have been identified to date. Repeated high-dose administration of triclosanwas also shown to change the ratio of these 2 metabolites in hamsters, mice, and monkeyswith the sulphate shown to predominate following chronic oral administration.

Triclosan was shown to be excreted primarily in the faeces of mice and rats following oraladministration, while the predominant excretion route in hamsters and monkeys was via theurine. The faecal excretion route also predominated in rats following dermal applications oftriclosan. In addition, evidence of enterohepatic circulation was found for rats and mice,based on autoradiography data, dual peak plasma concentrations, excretion data followingi.v. dosing, and bile absorption studies. However, the data available for hamsters do not

provide evidence for enterohepatic circulation in this species.

Some notable parallels and differences exist among the 3 rodent species, namely mice, rats,and hamsters, that are the most well-characterized species regarding triclosan nonclinicalpharmacokinetics. For a target single-dose level of 200 mg/kg body weight, the maximalblood concentrations of both sexes were 213 to 267, 153 to 183 (only males), and 359 to385 µg/g in mice, rats, and hamsters, respectively, with the resulting exposures for thisdose of 4,505 to 6,322, 3,237 (average for males) and 4,298 to 6,010 µg*hr/mL (Table23). Thus, in these single-dose studies, rats had the lowest blood levels, whereas mice andhamsters had comparable ranges in exposure levels. In contrast, in rodent chroniccarcinogenicity bioassays of at least 18 months duration (i.e., at least 18 months dailyexposure in the diet) using different target dose levels, plasma levels in mice were slightly

higher or comparable to plasma levels in rats (based on dose-normalized data) and muchhigher than plasma levels in hamsters (greater than 4- to 5-fold) (Table 29).

Evidence from pharmacokinetic studies suggests that the mouse liver is highly exposed totriclosan levels. Repeated dosing in mice led to an increased half-life for triclosan and itssulfate conjugate as measured in the kidney, and elevated liver triclosan levels (~2- to3-fold) in male mice (with respect to levels measured in the plasma and kidney) [van Dijk,1995 (98)]. The higher levels of triclosan in mouse liver than in plasma may be correlatedwith toxicology findings in mouse toxicology studies. Differences between mice, rats, andhamsters in hepatic levels of triclosan [van Dijk, 1994 (80); van Dijk, 1995 (81); van Dijk,1996 (82)] also may correlate with differences in the incidence of liver-related findings inthese species, with the mouse showing greater sensitivity to liver effects, but both the ratand hamster showing either a lower incidence or no liver effects (see Sections 3.3.5 and3.3.7).



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NOTEKinetic studies in HUMANS are described in section 3.3.11

3.3.10. Photo-induced toxicity

The data from these studies show that there is no evidence of photosensitisation in theguinea pig, mouse, and pig following exposure to triclosan. As in the case of thesensitisation studies in the guinea pig, these photosensitisation studies also were conductedprior to 1980, preceding the implementation of GLP regulations and OECD guidelines. In aphotosensitisation study in the guinea pig, the majority of animals in all dose groups,including negative controls showed an inflammatory response 4 hours post-irradiation withunfiltered light; by 24 hours, none of the animals in the triclosan (1% in solution) ornegative control groups showed an erythemic reaction [Thomann and Maurer, 1978 (7)]. Itshould be noted that a phototoxic response is expected with unfiltered light, as UVB ishighly cytotoxic; hence the positive response in the negative control animals. No positiveresponses in any dose groups were reported following irradiation with filtered light. Theauthors concluded that there was no indication of photosensitisation activity with triclosan inthe guinea pig. There was also no evidence of phototoxicity in the mouse or pig with

triclosan (0.1% in methanol, or 0.1 and 1.0% in petrolatum) as reported in another study;however, detailed results were lacking in this study report [Urbach, 1973 (101)]. Basedprimarily on the guinea pig data, with supporting evidence from the mouse and pig data,there appears to be no potential for photosensitisation with triclosan.

Table 33: Findings from Photosensitisation Studies with Triclosan

Species (Strain) Application Details Major Findings Reference,

GLP and OECD

Status

Guinea pig, Pirbrightwhite

1.0% triclosan in solution,0.1 mL single application.

Irradiation: unfiltered light(UV-A, UV-B), 5 minutes, orfiltered light (Pyrexfilters), 15minutes

Unfiltered light: The majority ofanimals including controls showed an

inflammatory response 4 hours post-irradiation in all dose groups. By 24hours, none of the animals in thetriclosan or negative control groupsshowed an erythemic reaction.Filtered light: No positive responses.

Thomann andMaurer, 1978

(7)Predates GLPand OECD

Mouse, AlbinoSkh:hair-less-1;Pig, Hanford Labcominiature swine

Triclosan (0.1% in methanol,0.1 and 1.0% in petrolatum)Irradiation sources: UVC;UVB; UVA, and UVB.

No evidence of phototoxicity wasobserved.

Urbach, 1973(101)


Summary of Photosensitisation Data

In summary, the results of these non-GLP studies indicate that there is no evidence forphotosensitisation with triclosan in various formulations and concentrations (up to 1% inpetrolatum) in the guinea pig, mouse, and pig.

3.3.11. Human data

Triclosan has been used in consumer products for over 30 years as an antibacterial agentand disinfectant. It is widely used in external-use, over-the-counter personal care productssuch as dentifrices, deodorants, soaps, creams, and lotions. Triclosan was approved for useat a level of 0.3% in cosmetics products in 1986 by the European Community CosmeticDirective. The world-wide population of consumers exposed to triclosan includes both adults

and children, with not-unexpected exposure in infants occurring via breast milk at low levels[Adolfsson-Erici et al ., 2002 (102), Allmyr et al., 2006 (103), Dayan, 2007 (104)].



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Studies on triclosan levels in human blood samples from Sweden [Allmyr et al., 2006 (103),Sandborgh-Englund et al. 2006 (116)] and from Australia [Allmyr et al. 2008 (AR1)] and arecent biomonitoring study with urine samples from the US [Calafat et al. 2008 (AR2)indicate widespread exposure of the general population to triclosan from various sources,including personal care products.

The following sections describe the general safety data for triclosan based on consumer useinformation and on results from clinical studies, the pharmacokinetics of triclosan in bothadults and children following single and repeated oral and/or dermal exposures, and theirritation, sensitisation, and photosensitisation data for triclosan as evaluated in clinicaltests. Estimates and findings of exposure levels to triclosan are presented in Sections3.3.11.2.6 (for infants) and 3.3.11.2.7 of this opinion (and in the discussion).

3.3.11.1 Human Safety/Tolerability Data

3.3.11.1.1 Safety Data from Consumer Use Information

Consumer use information such as listings of adverse events for cosmetic products

containing triclosan is generally unavailable.

No serious adverse events have been reported for triclosan-containing toothpaste.

The low number of reported non-serious adverse events for triclosan-containing toothpaste(frequency of 0.27 complaints/100,000 units sold) included reactions that were associatedwith the use of dentifrices in general, as identified during clinical testing (e.g., dry mouth,mouth irritation/burning, sensitive teeth, altered taste or oral sensation, exfoliation). Othernon-serious adverse events were those that represented a variety of much moreinfrequently reported effects (i.e., usually reported by only one or two consumers, resultingin a reporting frequency of 0.09 complaints/100,000 units sold). These rare non-seriousevents, which may not have been related to the use of the triclosan-containing toothpaste,

included stomach ache, belching, alopecia, anxiety, headache, black or coated tongue,dizziness, excess saliva, upper respiratory infection, increased urge to urinate, andshortness of breath.

Thus, based on consumer use information for triclosan-containing toothpaste, whichrepresents a wide-spread use that includes the potential for systemic exposure through theoral route, data indicate that triclosan can be used safely and with good tolerability at levelsthat also are found in personal care products.

3.3.11.1.2 Sa f e t y / T o l er a b i l i t y D a t a f r om Cl i n i c al St u d i e s

In addition to the indications of good tolerability and safety from historical and consumeruse of personal care products containing triclosan, a number of clinical studies haveinvestigated the safety and tolerability of triclosan.

Table 34: Findings from Human Safety Studies for Triclosan

No. Subjects Dosing Regimen Major Findings Reference

153 Twice daily brushing, for12 weeks, with 0.2%triclosan in toothpaste(75 subjects) orNaF/silica toothpaste(78 subjects).

For triglycerides, the triclosan group differed from theplacebo group (p<0.01) at 3 weeks, but not at 12weeks. There was no difference between treatmentand control groups for any of the other liver functiontests.

Colgate-Palmolive,1994(105)



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No. Subjects Dosing Regimen Major Findings Reference

112 Normal daily use of atoothpaste containing0.2% triclosan and 0.5%zinc citrate for 52weeks, followed by a 13-week washout period.

The data showed increases in white blood cells andlymphocytes throughout the study, with decreases inmean corpuscular haemoglobin/concentration.Biochemical analyses showed increases in sodium,cholesterol (total, LDL, and HDL), uric acid, andcreatinine and decreases in bilirubin at all time pointsin the study in either males or females or both sexes.These changes were not attributed to triclosan.

Safford, 1991(106)

112 (86subjectscompletedstudy)

Normal daily use(according to subject’snormal brushing habits)of a toothpastecontaining 0.2%triclosan and 0.5% zincfor 12 weeks, with anextension of the study to52 weeks (followed by a13-week observationperiod)

Blood samplings at baseline and 4, 12, 24, 38, 52,and 65 weeks. Standard haematology and clinicalbiochemistry results showed some statisticallysignificant changes periodically (compared to baselinevalues), but no pattern to the changes. Somesignificant correlations were found betweenparameters and triclosan blood levels, but these wereinconsistent over the course of the study in terms oftime or sex of subject. Therefore, these changeswere not attributed to triclosan toothpaste use. Oralirritation data including subjective “complaint level”for the whole trial period were summarised and found

to be similar to effects experienced in similar trialsinvolving toothpaste. There were no withdrawalsfrom the trial due to adverse reactions.

Barnes, 1991(107)

3,000 Normal dentifrice use,for 3 years, with silicondioxide-basedtoothpaste containing0.243% fluoride and0.3% triclosan, or0.243% NaF, or 0.331%NaF.

No treatment-related changes were reported for anyparameter measured, including haematology, clinicalchemistry, and urinalysis parameters. The datashows that the use of toothpaste containing 0.3%triclosan produces no adverse effects compared totriclosan-free toothpaste formulations.

Fishman,1994(108)

20 0, 1, 5, 9, 12, 15, 18,21, 24, 27, or 30 mgtriclosan in capsule,depending on studyphase. Single-dose andrepeat-dose dependingon study phase (up to30 days).

There were 6 dropouts (5 triclosan and 1 placebo)due to adverse effects (skin reactions - 3 subjectsincluding 1 placebo subject; increase in liver enzymes- 2 subjects; and, antrum gastritis - 1 subject).These findings were not considered to be attributableto triclosan, as they are not uncommon inpharmacological studies. Based on vital signs, ECG,lung function, neurological examination and mostlaboratory parameters, triclosan was considered to bewell-tolerated. There were slight increases in liverenzyme values including SGPT, SGOT, and gamma-GT.

Lucker et al., 1990(109)

Four human studies have evaluated the safety of triclosan in toothpaste products. Thesestudies tested triclosan-containing toothpaste preparations in study populations overperiods ranging from 12 weeks to 3 years at concentrations of 0.2% triclosan [Colgate-Palmolive, 1994 (105)], 0.2% triclosan with 0.5% zinc citrate [Safford, 1991 (106); Barnes,

1992 (107)], and 0.3% triclosan with 0.243% fluoride [Fishman, 1994 (108)]. Endpointsmeasured included blood chemistry and haematological parameters in all 4 studies. Inaddition, urinalysis parameters were evaluated in 1 study [Fishman, 1994 (108)]. In allstudies, there were no changes indicative of overt toxicity. Reported changes inhaematological and/or clinical chemistry parameters that did occur could not be attributedto the use of the triclosan-containing toothpaste.

In a separate study of pharmacokinetics and tolerability, consumption of escalating dailydoses of triclosan in capsule form (0 to 30 mg/capsule) by 20 volunteers was reported to bewithout overt effects on ophthalmic, neurologic, cardiac, and lung function evaluations[Lucker et al., 1990 (109)]. There were slight increases in liver enzyme values of thetreated subjects, but this is in contrast to the four human studies (described above) that

reported a lack of findings in liver enzyme parameters. Five of the treated participantsdropped out during the course of the study; however, the adverse events reported in thesesubjects could not be attributed to triclosan.



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One of the safety studies [Safford, 1991 (106)] reported on the concentrations of triclosanin the blood following the use of a toothpaste containing 0.2% triclosan and 0.5% zinccitrate. In this study of 112 participants, the average triclosan concentration in the blood of15.5 ng/mL was reported to rise to 31.2 ng/mL after a period of 4 weeks on study. Thisconcentration was reported to subsequently level off with further use of the triclosan-

containing dentifrice.

In summary, the human safety studies reviewed showed no signs of overt toxicity in over3,000 subjects that used triclosan-containing toothpaste for 12 weeks to 3 years.

3.3.11.2 Human Pharmacokinetics and Metabolism

A total of over 30 pharmacokinetic studies investigating the absorption, metabolism andexcretion of triclosan in humans have been reviewed. In these studies, several differentroutes of administration, including oral exposure to triclosan-containing products (e.g.,toothpaste), oral ingestion of capsules, aqueous solutions, and dental slurries (i.e., following

brushing with triclosan-containing toothpaste), and percutaneous exposure (in vivo and invitro) have been investigated. Overall, ingested triclosan is almost completely absorbed,whereas oral cavity and percutaneous exposure to triclosan-containing products (e.g.,toothpaste, soap, cream, etc .) results in limited absorption. Following all routes ofadministration, absorbed triclosan is nearly totally converted to glucuronic and sulphuricacid conjugates (varied relative proportions), with only trace amounts of the parentcompound detected in the plasma, and the predominant route of excretion for triclosan isthe urine, with the majority of the compound appearing as the glucuronide conjugate.

3.3.11.2.1 Pharmacokinetics

The pharmacokinetic parameters assessed for triclosan in humans include the Cmax, the time

required to reach Tmax, the AUC values for plasma concentrations versus time, and theelimination half-life (t½) of plasma concentrations. These parameters were assessedfollowing several different routes of administration, including oral exposure to triclosan-containing toothpaste (expelled dental slurry), and oral ingestion of triclosan-containingcapsules, aqueous solutions, and dental slurries (i.e., ingestion of dental slurry followingbrushing with triclosan-containing toothpaste). In general, the pharmacokinetic studiesreviewed herein analysed samples using HPLC or GC with electron capture detector.

The pharmacokinetic data for triclosan, as measured in children and adults following variousroutes of administration, are summarized in Table 35.

Table 35: Summary of Plasma Pharmacokinetics of Triclosan in Children and Adult Subjects

Parameters1

Route Subject Dose/DurationCmax

(ng/mL)Tmax (h)

AUC2 (ng*h/mL)

t½(h)

Reference

1 mg/single dose 23.3 5.00 208 13.4Oral(capsules)

Adults

15 mg/day for 36days

330.9 4.08 4,296 19.0

Lucker et al., 1990 (109)

10 mg/single dose 974.1 1.6 11,237 19.9 ConcordiaResearchLaboratories,1997a (110)

Oral (aqueoussolution)

Adults

4 mg/day for 21days

191.2 16.0 2,788 Colgate-Palmolive, 1989

(113)



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Parameters1

Route Subject Dose/DurationCmax

(ng/mL)Tmax (h)

AUC2 (ng*h/mL)

t½(h)

Reference

4 mg/single dose 218 1.5 2,600 21 Sandborgh-Englund et al.,2006 (116)

495.9 1.8 6,545 16.8 Colgate-Palmolive,1997a (111)

Children3 3.0 mg/singledose

N/A N/A N/A 12.7 ConcordiaResearchLaboratories,1997b (112)

6 mg/single dose 574.2 3.0 7,235 20.0 ConcordiaResearchLaboratories,1997a (110)

Oral (dentalslurry)

Adults

18 mg/day for 14days (dental

slurry swallowedafter brushing)

878.0 N/A 218,856 N/A BIBRAInternational,

1997 (114)

4 mg/day for 21days

26.7 12.0 329 N/A Colgate-Palmolive, 1989(113)

18 mg/day for 14days (dentalslurry expelledafter brushing)

145.5 N/A 34,855 N/A BIBRAInternational,1997 (114)

3.75 mg/singledose

242.9 4.0 2,818 14.6 Colgate-Palmolive,1997b (115)

Oral(toothpaste)

Adults

11.25 mg/day for12 days (3.75 mgx 3 brushingsdaily)

384.04,5 2.0 2,8446 N/A Colgate-Palmolive,1997b (115)

N/A, not available1 Cmax =maximal concentrations in blood and tissues, Tmax =the time required to reach the peak concentration,AUC= the plasma area under the concentration-time curve, T1/2= the elimination half-life of the radioactivity2 When both AUC(0

∞) and AUC(0

24h) values were reported, the former was used.

3 Children ranged from 8 to 12 years of age. Due to limited blood collection intervals in the study, limited or noCmax and Tmax data were available, and no AUC values were calculated for the available data [Concordia ResearchLaboratories, 1997b (112)].4 Sampling was conducted following the first of 3 brushings on Day 12 of the multiple-dose phase of the study.5 Plasma concentrations ranged from 353 to 384 between hours 1 to 7 of the study.6 AUC value is dose-normalized (mean AUC(0

24) corrected for number of brushings on that day (AUC24 /3)

As outlined in Table 35, Cmax and AUC values obtained from adult subjects generally

increased with increasing dose levels, with Cmax values ranging from 23.3 to 974.1 ng/mL,and AUC values ranging from 208 to 11,237 ng*h/mL for single doses ranging from1.0 mg/dose to 10 mg/day (corresponding to approximately 0.017 and 0.17 mg/kg bodyweight, respectively, for a 60 kg adult). For multiple doses ranging from 4 to 18 mg/day(corresponding to 0.067 and 0.30 mg/kg body weight/day for a 60 kg adult), Cmax valuesranged from 26.7 to 878 ng/mL, and AUC values ranged from 329 to 21,8856 ng*h/mL.Corresponding Tmax values ranged from 1.5 to 5.0 hours and from 4.08 to 16.0 hours forsingle and multiple doses, respectively. t½ values obtained from adult subjects remainedfairly constant across all the studies reviewed, ranging from 13.4 to 21.0 hours.

There are limited pharmacokinetic data for children, and no direct comparisons to adultswere possible, given differences in doses and dosing formulations in all of the studies, with

the exception that elimination was determined to be essentially the same for children andadults. Two single oral dose (3.0 mg triclosan from aqueous solution) studies were



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conducted in children [n = 11 children, aged 8 to 12 years in Colgate-Palmolive, 1997a(111) and n = 4 children, aged 9 to 12 years in Concordia Research Laboratories, 1997b(112)]. The mean elimination rate constants (Kel) for each study were calculated to be0.0453 h-1 (n = 11) (3) and 0.062 h-1 (n = 4) [Concordia Research Laboratories, 1997b(112)]. These values are similar to those calculated in adult subjects exposed to single oraldoses of triclosan from aqueous solution (Kel = 0.043 h-1), dental slurry (Kel = 0.043 h-1)

and toothpaste use (Kel = 0.0742 h-1) [Concordia Research Laboratories, 1997a (110)].Thus, the rate of elimination is comparable in children and adults.

Pharmacokinetic parameters have also been assessed in the saliva of adult subjectsexposed to 4 mg triclosan from toothpaste (not outlined in Table 35). Throughout the 2-hour period following tooth brushing, mean saliva triclosan concentrations and Ke and t½values revealed first-order kinetics [Gilbert, 1987 (117)].

Intra-subject comparisons were made following single and multiple oral doses of triclosan[Lucker et al., 1990 (109), Colgate-Palmolive, 1997b (115)], and following ingestion oftriclosan-containing dental slurry and triclosan aqueous solution [Concordia ResearchLaboratories, 1997a (110)]. The mean AUC values for the single- and dose-normalized

multiple-dose exposures were not different (dose-normalized AUC values for multiple-doseexposures not shown in Table 35), suggesting a linear disposition for triclosan afterrepeated brushing [Colgate-Palmolive, 1997b (115)]. Dose-normalized AUC and Cmax valueswere similar following ingestion of triclosan-containing dental slurry and triclosan aqueoussolution, suggesting that each route of administration results in similar levels of exposure totriclosan [Concordia Research Laboratories, 1997a (110)].

To investigate differences in exposure levels following different formulae of administration,inter-subject comparisons were made between triclosan-containing toothpaste (dental slurryexpelled) use versus ingestion of the dental slurry [BIBRA International, 1997 (114)], andbetween triclosan-containing toothpaste (dental slurry expelled) use versus ingestion oftriclosan aqueous solution [Colgate-Palmolive, 1989 (113)]. As would be expected, based

on AUC and Cmax values, ingestion of triclosan aqueous solutions (at levels simulating themaximum absorption of triclosan that would be possible from a triclosan-containingtoothpaste) resulted in higher levels of exposure to triclosan compared with the use oftriclosan-containing toothpaste (expectorated) [Colgate-Palmolive, 1989 (113)]. Similarly,higher AUC and Cmax values were observed in subjects who ingested the dental slurrycompared with those who expelled the slurry following brushing with triclosan-containingtoothpaste [BIBRA International, 1997 (114)]. For each study, there were no differencesbetween groups with respect to time to reach steady-state plasma triclosan concentrations[4 days in the 14-day study (BIBRA International, 1997 (114), and approximately 14 daysin the 21-day study (Colgate-Palmolive, 1989 (113)].

In summary, AUC and Cmax values appear to increase with increasing doses of triclosan.There does not appear to be a correlation between the duration of triclosan exposure(measured in days) from tooth brushing and the levels of exposure ( i.e., AUC values);however, the formula of administration does appear to affect both AUC and Cmax values.While the ingestion of triclosan-containing dental slurry and triclosan aqueous solution eachresults in similar levels of exposure to triclosan, both routes of exposure result in higherlevels when compared with the use of triclosan-containing toothpaste followed byexpectoration. These data show that there is limited absorption under normal conditions oftoothpaste use (i.e., expectoration and rinsing) compared with oral ingestion of triclosan-containing solutions (see absorption section).

Bioaccumulation/Bioretention

In the absence of data showing tissue levels of triclosan following single and repeated

exposures, evidence of a lack of bioaccumulation or bioretention of triclosan is provided byan examination and comparison of plasma triclosan levels in a number of studies.



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The plasma AUC0-inf level of triclosan was 2,818 ng*h/mL following single use of dentifricecontaining 0.3% triclosan (3.75 mg dose, with ingestion of the dentifrice) in a study of 21adult subjects [Colgate-Palmolive, 1997b (115)] (see Table 35). In the repeated dose phaseof the same study, subjects brushed 3 times daily for 12 days with ingestion of the dentalslurry (3.75 mg/dose). After 12 days of brushing, the plasma AUC levels following a single

dose was calculated to be 2,844 ng*h/mL after normalizing for the number of doses (AUC 0-

24h = 8,833 ng*h/mL per 3 doses per 24 hours). There was no significant differencebetween the single- and repeated-dose AUC values, suggesting a complete elimination ofthe daily triclosan dose, and no increase in the triclosan level during repeatedbrushing/ingestion.

Additional supporting evidence for a lack of bioaccumulation or bioretention of triclosan isshown in several studies in which there was no further increase in plasma triclosan levelsonce steady-state blood concentrations had been reached. Triclosan levels in plasma werecomparable from Days 7 to 21 in a study in which subjects either ingested 20 mL of anaqueous solution containing 0.01% triclosan or brushed twice daily with 1 g of a dentifricecontaining 0.2% triclosan (with expectoration and rinsing) (plasma levels ranged from 15 to

21 ng/mL in the dentifrice group) [Colgate-Palmolive, 1989 (113)]. In a longer-termparallel, double-blind 12-week study with dentifrice containing 0.2% triclosan, bloodsamples showed comparable mean levels of 16 and 14 ng/mL at 3 and 12 weeks,respectively [Lin, 1989 (135)]. In a 52-week tooth brushing study using dentifricecontaining 0.2% triclosan, total triclosan levels in plasma were consistently in the range of27 to 40 ng/mL from 4 weeks to the end of the 52-week exposure period [Safford, 1991(106)]. Altogether, the data from these 3 studies show consistency in plasma triclosanlevels following the use of dentifrice containing 0.2% triclosan. In a review of the data fromColgate-Palmolive, 1989 (113), Lin, 1989 (135), and Colgate-Palmolive, 1997b (115), thisconclusion was extended to dentifrices containing triclosan at levels up to 0.3% [Bagley andLin, 2000 (142)]. In addition, the data suggest that there is no accumulation of triclosanlevels as reflected in comparable plasma levels over the time course of each study,

suggesting a lack of evidence of bioaccumulation of triclosan.

In the absence of studies examining tissue concentrations over time, relatively invariableplasma concentrations of triclosan provide evidence of a lack of bioaccumulation followingdermal application in human studies. Plasma levels of total triclosan ranged between 85 and101 ng/mL between Days 5 to 20 in males and 41 to 47 ng/mL in females over the sametime period in which triclosan exposure occurred through the use of hand wash containing1% triclosan [Ciba Specialty Chemicals, 2002 (134)]. These data suggest a balancebetween absorption and elimination and a lack of bioaccumulation following dermalabsorption.

In summary, plasma AUC data for triclosan following either single or repeated oral dosingindicate a lack of accumulation of triclosan. The blood or plasma AUC is a measurement ofexposure to a given dose of drug or substance, encompassing both amount absorbed andamount eliminated. If the AUC of a 24-hour dose interval in a continuous dosing regimen isequal to the AUC of an equivalent single dose, the data would indicate a lack ofaccumulation (i.e., the complete elimination of the daily triclosan dose, with the absorptionequal to the elimination from the body in a given single-dose interval). In addition, thesteady and relatively invariable plasma levels of triclosan in long-term dosing (brushing)studies and in dermal application studies further suggest that triclosan does not accumulatein the body.

3.3.11.2.2 Absorption

The absorption of triclosan was measured following several different routes of

administration, including oral exposure to triclosan-containing products (e.g., toothpaste),



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oral ingestion of capsules, aqueous solutions, and dental slurries ( i.e., following brushingwith triclosan-containing toothpaste), and percutaneous exposure (in vivo and in vitro).

Absorption Following Oral Administration

Several studies of oral absorption have been conducted for exposures via capsules,

solutions, dental slurries, mouthwash, or toothpaste. Overall, triclosan administered via theoral route in capsules is almost completely absorbed, as indicated by urinary eliminationdata [Lucker et al., 1990 (109); Stierlin, 1972b (121)]. Following exposure to 15 mg/day oftriclosan (corresponding to 0.25 mg/kg body weight/day) for 36 days, 90% of theadministered dose was eliminated in the faeces (10%) and urine (80%), as shown in anintra-subject study [Lucker et al., 1990 (109)]. Similarly, more than 98% of theadministered dose was recovered in the urine (87%) and faeces (11%) following a single200.5 mg dose of radiolabelled triclosan in a gelatine capsule (corresponding to 3.3 mg/kgbody weight) [Stierlin, 1972b (121)].

Two separate studies examined the effects of the formulation of the administered triclosanon oral absorption [Concordia Research Laboratories, 1997a (110); Colgate-Palmolive, 1989

(113)]. A study comparing oral ingestion of triclosan from dental slurry (following brushingwith triclosan-containing toothpaste) with oral ingestion of triclosan from aqueous solutionrevealed that the onset and rate of absorption of triclosan was faster for the aqueoussolution compared with the dental slurry [Concordia Research Laboratories, 1997a (110)].Results from a study comparing triclosan-containing toothpaste use (expectoration of dentalslurry) with ingestion of triclosan aqueous solution confirmed that the amount of triclosanabsorbed from normal toothpaste use (including expectoration and rinsing) is extremely low(i.e., 5 to 10% of the dose, which corresponds to 9 to 14% of the amount absorbed andexcreted following ingestion of an equivalent amount of triclosan in aqueous solution) due todecreased ingestion [Colgate-Palmolive, 1989 (113)]. The results of these studies show thatnormal toothpaste use would be expected to result in low levels of total absorption togetherwith a slow onset and rate of absorption compared with oral ingestion of triclosan in an

aqueous solution.

Oral retention of triclosan following the use of triclosan-containing products (toothpaste andmouth rinse) was examined in 2 studies [Gilbert, 1987 (117); Lin, 2000 (118)]. In onestudy, saliva samples were collected after the first of two daily brushings with triclosan-containing toothpaste (2 mg triclosan per brushing). Mouth rinse samples were collectedfollowing the use of a mouth rinse formulated to recover triclosan after the second brushing.The results indicated that approximately 25% of the triclosan dose is retained in the mouthfollowing tooth brushing, with the remainder being recovered on the toothbrush,expectorated and rinsed out. The use of a non-triclosan mouth rinse following brushingdecreases oral retention further, with approximately 14% of the retained triclosan (i.e., ofthe 25%) being recovered by the mouth rinse [Gilbert, 1987 (117)]. In a separate study,4.5 mg of triclosan was administered in a mouth rinse twice daily for 21 days. Oralretention of triclosan was measured to be 4 to 13% of the daily dose, and buccal absorptionof triclosan was estimated to be 2 to 4% of the daily dose [Lin, 2000 (118)].

Absorption Following Percutaneous Administration

The main findings from in vivo and in vitro percutaneous absorption studies for triclosan aresummarized in Table 36, with discussion in the paragraphs that follow.

Table 36: Findings from Human In Vivo and In Vitro Percutaneous Absorption Studies forTriclosan

Subject Method Major Findings Reference

In Vitro Studies



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In vitro Breast or abdominalskin (0.64 cm2) wasobtained and exposedto a tritiated triclosansolution for up to 24hours.

Non-GLP. After 24 hours, approximately 6.3% ofthe applied dose appeared in the receptor fluid, and46%, 24%, and 22% remained on the skin surface,in the epidermis and dermis, and stratum corneum,respectively. Of the radioactivity in the receptorfluid at 24 hours, 0.8% of the applied dose waspresent as triclosan, 3.5% as triclosan glucuronide,and 0.2% as triclosan sulfate. Of the radioactivity inthe skin at 24 hours, 12% of the dose was recoveredas triclosan, 3% as triclosan glucuronide, and 3% astriclosan sulfate.

Moss et al .,2000(21)

In vitro Female epidermal skinsamples from cosmeticsurgery, mounted indiffusion cells; singleapplication of 0.2%triclosan in w/oemulsion; leave-on.

GLP-compliant. Conducted comparable to OECDGuideline No. 428. After 24 h, 95% recovery of theapplied dose of triclosan. Period of penetration wasbetween 8 and 24 h following a lag phase ofapproximately 8 h. The rate of skin permeationreached ~0.008 µg/cm2 /h. After 24 h, 3.9±0.4% ofthe applied dose of triclosan had penetrated into thereceptor fluid (0.14±0.01 µg/cm2). As measured at24 h, triclosan in the surface material was 76% of

the applied dose (65% in the 24-h surface wipe and11% in the first 3 tape strips of skin). Succeedingtape-strips (strips 4-20) contained 6.8% of theapplied dose (0.25 µg/cm2), and 7.4% of the dosewas recovered from the remainder of the sample ofskin (0.28±0.05 µg/cm2). Percutaneous absorptionwas calculated to be 11.3% (0.42 µg/cm2) based onreceptor fluid plus remainder of skin after removal oftape strips 1-20.

Ciba SpecialtyChemicals,1998a (130)

In vitro Female epidermal skinsamples from cosmeticsurgery mounted indiffusion cells; singleapplication ofdishwashing liquid

(0.2% triclosan); rinse-off after 30 minutes.

GLP-compliant. Conducted comparable to OECDGuideline No. 428. After 24 h, there was 89%recovery of the applied dose of triclosan. Period ofpenetration was between 2 and 6 h following a lagtime of about 2 h. The rate of skin permeationreached ~0.01 µg/cm2 /h. After 24 h, 2.3±0.3% of

the applied dose had penetrated into the receptorfluid (0.093±0.01 µg/cm2). As measured at 24 h,triclosan in the surface material was 4.3% of theapplied dose (<1% of wiped off surface); 70% hadbeen recovered in the 30-minute rinsate.Succeeding tape-strips (strips 4-20) contained 3.0%of the applied dose (0.25 µg/cm2), and 9.7±1.7% ofthe dose was recovered from the remainder of thesample of skin (0.39±0.06 µg/cm2). Percutaneousabsorption was calculated to be 12.0% (0.483µg/cm2) based on receptor fluid plus remainder ofskin after removal of tape strips 1-20.

Ciba SpecialtyChemicals,1998b (131)

In vitro Female epidermal skinsamples from cosmeticsurgery mounted in

diffusion cells; singleapplication of adeodorant formulation(0.2% triclosan); leave-on.

GLP-compliant. Conducted comparable to OECDGuideline No. 428. After 24 h, 84% recovery of theapplied dose of triclosan. Period of penetration was

between 8 and 24 h following a lag phase of 6-8 h.The rate of skin permeation reached ~0.002µg/cm2 /h. After 24 h, 0.85±0.13% of the applieddose of triclosan had penetrated into the receptorfluid (0.033±0.006 µg/cm2). As measured at 24 h,triclosan in the surface material was 64% of theapplied dose (40% in the 24-h surface wipe and24% in the first 3 tape strips of skin). Succeedingtape-strips (strips 4-20) contained 13.2% of theapplied dose (0.50 µg/cm2), and 6.8% of the dosewas recovered from the remainder of the sample ofskin (0.27±0.09 µg/cm2). Percutaneous absorptionwas calculated to be 7.65% (0.303 µg/cm2) based onreceptor fluid plus remainder of skin after removal oftape strips 1-20.

Ciba SpecialtyChemicals,1998c (132)



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In vitro Female epidermal skinsamples from cosmeticsurgery, mounted indiffusion cells; singleapplication of a soapsolution (0.02%triclosan); rinse off after10 minutes.

GLP-compliant. Conducted comparable to OECDGuideline No. 428. After 24 h, there was 91%recovery of the applied dose of triclosan. Period ofpenetration was between 2 and 6 h (following ashort lag time of <1 h). The rate of skin permeationreached ~0.001 µg/cm2 /h between 2 and 6 hours.There was marked decrease in penetration between6 and 8 h, reaching a plateau by 24 hours. After 24h, 2.3±0.25% of the applied dose of triclosan hadpenetrated into the receptor fluid (0.0096±0.0011µg/cm2). As measured at 24 h, triclosan in thesurface material (with stratum corneum) was 9%.Succeeding tape-strips (strips 4-20) contained 4.3%of the applied dose (0.018 µg/cm2) and 4.9%(0.021±0.0042 µg/cm2) of the dose was recoveredfrom the remainder of the sample of skin.Percutaneous absorption was calculated to be 7.2%(0.0306 µg/cm2) based on receptor fluid plusremainder of skin after removal of tape strips 1-20.

Ciba SpecialtyChemicals,1998d (133)

In vitro Single versus 6

applications in 3 days of0.25 mL of an 8% (w/v)conventional soap(freshly prepared orequilibrated over 1week) or non-soapdetergent suspension(freshly prepared) eachcontaining 0.08% (w/v)[3H]DP300 was appliedto the lower back (20cm2) for 2 minutes.

Pre-dates GLP. Autoradiography of skin at 10

minutes after a single application of the various[3H]DP300 soap suspensions showed very low silvergrain densities on the stratum corneum and in theepidermis, low or very low densities in the upperdermis, and very low or nil densities in the lowerdermis. No grains were seen in the follicles orsebaceous glands. At 48 h after the singleapplication, no silver grains were seen, except in thecorneum after application of the fresh soappreparation. Scintillation counting showed nosignificant differences between the soap vehicles orin the single vs. repeated applications.

Black et al.,

1975(27)

In Vivo Studies, Percutaneous Absorption Only

3 subjects (2treatment, 1control)

10.0g soap containing0.75% Irgasan® DP300 used for full bodybathing for 75 days.

Blood levels reached a plateau (7.0 to 19.1 ng/mL)immediately (i.e., within 2 hours following first bath)and did not accumulate throughout study.

Ciba-Geigy,1972a(119)

125 maleand female

21-day hand washingstudy with dilutedderma-san containing0.25% Irgasan® DP300 or scrub containing1.0% Irgasan® DP 300.7-day withdrawalperiod.

Blood levels increased as duration of study increasedfor 0.25% (28 to 68 ng/mL) but not 1.0% Irgasan®DP 300 (87 to 94 ng/mL). Dose-dependent increasein blood levels was observed. Blood levels return tonear baseline (16 ng/mL, baseline = <10 ng/mL) bythe end of withdrawal period. Irgasan was presentin the plasma in a conjugated form (not specified):no free Irgasan detected.

Ciba-Geigy,1973b(120)

2 females 0.34 and 0.51 mgradiolabelled GP 41535/kg body weightapplied to area of skin

measuring 8 x 8 cm andcovered for 24 h withocclusive dressing(1 dose/subject).

Only a very small percentage of the administereddose was absorbed and was excreted completely; 2to 7% was recovered in the urine, 0.5 to 2% in thefaeces and the remainder in the dressing. The blood

concentrations of GP 41 353 were ≤0.01 µg/mL atall time points measured (1, 3, 5, 9 and 24 h afterapplication).

Stierlin, 1972b(121)

9 males Group of 3 subjectsreceived singleintravenous dose of14C-irgasan® CH3565;group of 6 subjectsreceived singleapplication (52 µg) of14C-irgasan® CH3565suspended in Ivory soapto 13 cm2 area offorearm for 24 h.

Following intravenous administration, the majority ofCH 3565 was accounted for in the urine(65.4%±13.5% of the injected dose) and faeces(20.6%±10.4%). The half -life of CH 3565 wasapproximately 10 h.

Following percutaneous application, an average of8.9%±3.2% of the CH 3565 dose was absorbedpercutaneously.

Maibach, 1969(123)



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1 female and3 male

10 g of surgical scrubproduct containing0.75% by weight ofIrgasan® DP 300 wasapplied to the surfacearea of both hands ofeach subject (2washings/day for 3minutes each for a totalof 2 weeks).

The amount of DP-300 in blood increasedimmediately after washing, reaching a plateau at the15 ng/mL level in the blood after the fourth day ofhand washing (8 washes). 24 h afterdiscontinuation of the hand washing, 2 subjects hadno detectable amounts of Irgasan® DP 300 in theblood and by 48 h after discontinuation the third hadno detectable amounts of Irgasan® DP 300 (1subject dropped out of the study due to illness).Therefore, small amounts of Irgasan® DP 300 areabsorbed through the skin and blood levels rapidlydrop upon discontinuation of application.

Ciba-Geigy,1972b(124)

6 male Single dermalapplication of 5 g ofCGP. 433 cream(equivalent to a dose of150 mg triclosan).Applied to back of eachsubject (surface area =900 cm2).

Urinary excretion of free triclosan ranged from 0.006to 0.041% of the administered dose during the 48-hurine collection period. Urinary excretion of freeplus glucuronide conjugated triclosan ranged from2.52 to 6.47% of the administered dose during the48-h urine collection period. Results indicate thattriclosan is slightly absorbed percutaneously.

Caudal et al .,1974 (125)

6 subjects 3 different soapformulations (Ivory, Dialbase and Colgate basesoap), each with andwithout 2% solution ofCH3565 were containedin glass boats. Eachsubject (2 sets ofsubjects) had a glassboat taped to eachforearm (one withCH3565, the otherwithout). Theapplications covered asurface area of 15 cm2

for 6 h (singleapplication).

Analysis of the remaining contents of the boats andthe washings from the skin revealed 100% recoveryof 2% CH3565 from all 3 soap formulations for thefirst set of subjects. For the second set of subjects,recovery was 96% for the Ivory and Colgate soapbases. The computed average recovery of eachformulation was 98%, suggesting that an average of2% of the CH3565 was absorbed.

Schenkel andFuria, 1965(126)

4 subjects Total of 10 mL ofApprove® skin cleanser(containing 0.75%triclosan) lathered onhands and forearms fora total of 8 minutes(single application).

Absorption of triclosan was minimal and occurredover a period of 8 h, at which time peak plasmaconcentrations of free plus conjugated triclosanranged from 15 to 31 ng/mL. Plasma concentrationsof free triclosan ranged from <3 to 4 ng/mL. Meantotal urinary excretion of free plus conjugatedtriclosan was 627±101 µg over a period of 48 hours.Urine concentrations of free triclosan ranged from<3 to 16 ng/mL.

Thompson et

al ., 1975a(127)

4 subjects Application of 1.0 mL of0.5% GP 41353 PatientSkin Prep (equivalent to

5 mg of triclosan) ontoa 100 cm2 area ofnormal and abradedabdominal skin, theformer with and withoutan occlusive dressing(three 12-hourapplications, separatedby 4 weeks).

In the absence of an occlusive dressing theabsorption of triclosan was below the limit ofdetection (plasma levels <15 ng/mL). The presence

of the occlusive dressing enhanced absorption(plasma levels of free plus conjugated triclosan 112to 192 ng/mL 4 to 8 h after application, declinedslowly over 32 to 96 hours). Urinary excretion offree plus conjugated triclosan accounted for 6 to14% of the dose without occlusive dressing, and 40to 58% of the dose with occlusive dressing. Thesevalues decreased to <2% by 72 to 96 h afterapplication. Absorption was not markedly increasedby abrasion by application and removal of cellulosetape.

Thompson et

al., 1975b(128)



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Single: 6males8-day: 6males (4Caucasian, 2AfricanAmerican);31-day: 11males (5Caucasian, 6AfricanAmerican,including the2 from the 8-day)

Single versus 8-day (22applications) versus 31-day (91 applications)scrub study. 10 mL ofGP 41353 Surgical HandScrub containing 50 mgtriclosan applied tohands and forearms per5-minute application.

Single: 10 to 24 hours following single application,maximum plasma concentration of free plusconjugated triclosan reached 62 to 143 ng/mL andreturned to baseline over a period of 56 to 96 h. 2.6to 6.6% of the applied dose was excreted in theurine as free plus conjugated triclosan (eliminationhalf-life 14.4 to 38.4 h).8-day: Plasma concentration of free plus conjugatedtriclosan still increasing on Day 8 (maximumconcentrations ranging from 490 to 715 ng/mL andelimination half-lives ranging from 1.4 to 2.1 d forCaucasian subjects; maximum concentrationsranging from 1,640 to 1,780 ng/mL and eliminationhalf-lives ranging from 11.3 to 15.6 d for AfricanAmerican subjects). Plasma concentrations of freetriclosan ranged from <3 to 13 ng/mL for allsubjects.

31-day: maximum plasma concentration of free plusconjugated triclosan (740 to 1,030 ng/mL) wasreached at Days 12 to 15 for Caucasians. Plasma

concentration of free triclosan was 16 and 6 ng/mLon Days 12 and 19, respectively. African Americansubjects were categorized as fast- or slow-eliminators. Slow eliminators had maximum plasmaconcentrations of free plus conjugated triclosanranging from 3,400 to 4,080 ng/mL and baselinevalues were still not attained by Day 78. The fast-eliminators had max concentrations ranging from554 to 690 ng/mL and baseline values were attainedby Day 50. Plasma concentrations of free triclosanfor slow- and fast- eliminators, respectively, were<3 and 9 ng/mL on Day 12 and <3 and 10 on Day19.

Thompson etal ., 1976 (129)(single versus 8-day)Thompson,1975(122)(8-day versus 31-day)

6 healthymales, 15leukaemiapatients,additional 4subjects

Healthy males used 1%DP300 soap bar for 11months. Leukaemiapatients bathed dailywith 1% DP300 soapbar for 5 weeks. 4subjects applied anaerosol anti-perspirantcontaining 0.1% DP300daily for 4 weeks.

DP300 was not readily absorbed through the skinfollowing repeated topical application of hygienicproducts. Maximum blood levels of total triclosanwere 44, 500, and 18 ng/mL for healthy subjectsusing soap bar, leukemic subjects using soap bar,and subjects using anti-perspirant, respectively.Blood levels of free triclosan did not exceed 8 ng/mLfor healthy subjects using soap bar, or subjectsusing anti-perspirant. Leukemic subjects using soapbar had blood levels of free triclosan as high as 500ng/mL. Maximum urine levels of total triclosan were1,106, >1,001, and 890 ng/mL for healthy subjectsusing soap bar, leukemic subjects using soap bar,and subjects using anti-perspirant, respectively.

Hong et al .,1976(24)

7 healthymales and 6

healthyfemalescompletedstudy

Volunteers washed theirhands with ~3-5 mL of

the test material(commercial hand washcontaining 1% triclosan)6 times/day, ~every 2 hduring the day, for alathering time of 15seconds/washingfollowed by thoroughrinsing, for 20consecutive days.

Steady-state plasma levels of free and total (free +conjugated) triclosan were measured. Only about

10% of total triclosan was present as free(unconjugated) triclosan. Total plasma triclosanlevels were consistently higher in males thanfemales. At steady-state (on Day 20), triclosanlevels in plasma were: 95.2 ng/mL in males and47.4 ng/mL in females; 73.1 ng/mL for both sexescombined. Steady-state plasma levels wereestimated to have been reached by Day 7. Plasmalevels rapidly decreased after cessation of handwashing. Elimination half-life:1.4 d. There wasconsiderable inter-individual variability in plasmalevels. Investigators concluded that use of the 1%hand wash formulation resulted in low levels ofsystemic exposure (lower than oral route).

Ciba SpecialtyChemicals,

2002 (134)

In Vivo Studies, Combined Oral and Percutaneous Absorption



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20volunteers(10/group)

Group 1: placebo soap,talc, antiperspirant +triclosan-containingtoothpaste (0.215%).Group 2: triclosan-containing soap, talc,antiperspirant(concentrations oftriclosan not specified)+ triclosan-containingtoothpaste (0.215%).Exposure for up to 56 d.

Total triclosan plasma levels did not generallyexceed 100 ng/mL, and levels quickly diminishedbelow detection level upon cessation of treatment(i.e., by Day 64). The amount of unconjugatedtriclosan levels in all samples was <10 ng/mL.Triclosan was present in plasma mainly as itsglucuronide-conjugated metabolite. Wide inter-subject variation prevented meaningful comparisonsbetween groups 1 and 2.

BIBRAInternational,1988(137)

68Caucasian,54 African-Americans,and 45 Asiansubjects, 18-65 years

Group 1: placebo soapand underarmdeodorant + triclosan-containing toothpaste(0.28%). Group 2:soap (0.75%, 2X/day),underarm deodorant(0.39% 1X/day) +

triclosan-containingtoothpaste (0.28%).Daily exposure for 3months.

Plasma levels of triclosan at Weeks 3, 6, and 13showed slight, significantly increased blood levels oftotal triclosan and of glucuronide-conjugatedtriclosan in Group 2 (triclosan-containing toothpasteplus triclosan-containing hygiene products)compared to Group 1 (triclosan-containingtoothpaste alone) [e.g., 23.79 vs. 18.99 ng/mL totaltriclosan at Week 13]. Note: sulfate-conjugated

triclosan levels were difficult to interpret due toinefficiencies in the analysis. These data showpercutaneous absorption of triclosan from personalhygiene products.

Beiswanger andTuohy, 1990(138)

Absorption Following I n V i t r o Percutaneous Administration

3H-labelled triclosan has been used to examine percutaneous absorption in a number of invitro studies. Percutaneous penetration of 30.3% of the total applied dose of triclosan in anethanol/water solution was measured 24 hours after application of the dose to human skinsamples in a diffusion cell system [Moss et al ., 2000 (21)]. Results from studies using skinsamples in diffusion cells showed limited percutaneous absorption following application of

triclosan to the skin surface in any of the formulations used. The amount of triclosan testedin most of these studies (0.2%) is within the range of anticipated concentrations inconsumer products (from 0.15 to 0.3%). The low concentration in the soap solution study(0.02%) was intended to simulate actual-use conditions (i.e., a soap solution of 0.2%triclosan would be diluted with water when applied to the skin). Appropriate rinse stepswere included in the rinse-off product studies. As recommended by SCCP guidelines (SCCP,2006), calculations of dermal absorption included the amounts of triclosan recovered in thedermis and epidermis layers (i.e., the “remaining sample of skin” after removing thestratum corneum, represented by tape strips 1 to 20 in these studies) and the amountrecovered in the receptor fluid. Table 37 presents a summary of the values from the in vitro studies conducted in human skin samples. The results from the autoradiography study[Black et al ., 1975 (27)] indicated no significant differences in the low levels of

percutaneous absorption between different soap vehicles or between single versus multipleapplications [Black et al ., 1975 (27)]. Overall, the dermal absorption of triclosan was shownin these in vitro studies to be lower in human skin than in rat (7.2 to 30.3% versus 41.2%in studies in rats, including the data from the study using ethanol/water formulation)].

Table 37: Summary of Dermal Absorption Values in Human Skin Samples from In Vitro Studies for Triclosan

Dermal Absorption

(SCCP)1

Test Formulation % Triclosan in test

material

% µg/cm2

Reference

w/o Emulsion 0.2% 11.3 0.420 Ciba Specialty Chemicals,

1998a (130)Dishwashing 0.2% 12.0 0.483 Ciba Specialty Chemicals,



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Dermal Absorption(SCCP)1

Test Formulation % Triclosan in testmaterial

% µg/cm2

Reference

formulation 1998b (131)

Deodorant formulation 0.2% 7.7 0.303 Ciba Specialty Chemicals,1998c (132)

Soap solution 0.02% 7.2 0.0306 Ciba Specialty Chemicals,1998d (133)

Ethanol/waterformulation

-- 30.3% -- Moss et al., 2000 (21)

1 Dermal absorption = amount measured in the dermis, epidermis (without stratum corneum) and the receptorfluid (SCCP, 2006). Thus, for data from the in vitro studies, dermal absorption = “remainder of skin” + receptorfluid

Absorption Following I n V iv o Percutaneous Administration

The in vivo percutaneous absorption of triclosan following single applications of triclosan-

containing products (e.g., cream and soap) has been consistently reported as only a smallproportion of the applied dose (i.e., generally ≤9%) [Stierlin, 1972b (121); Maibach, 1969(123); Caudal et al., 1974 (125); Schenkel and Furia, 1965 (126); Thompson et al ., 1975a(127)].

Additional studies were conducted to determine blood and urine levels of triclosan (as ameasure of absorption) following single and multiple percutaneous applications of triclosanin different vehicles including soaps, creams, other skin cleansers and surgical scrubs.Together, these data support findings indicating that percutaneous triclosan absorption isminimal [Caudal et al., 1974 (125); Thompson et al ., 1975a (127); Hong et al., 1976 (24)].In general, blood levels of triclosan increased immediately after percutaneous application[Ciba-Geigy, 1972a (119); Ciba-Geigy, 1972b (124)], were enhanced by the presence of an

occlusive dressing [Thompson et al ., 1975b (128)], and were proportional to the doseapplied [Ciba-Geigy, 1973b (120)]. Percutaneous absorption of triclosan from the use oftriclosan-containing soap, underarm product and talc was also shown to be detectable insubjects already using triclosan-containing toothpaste [BIBRA International, 1988 (137);Beiswanger and Tuohy, 1990 (138)].

3.3.11.2.3 Metabolism

The metabolism of triclosan was investigated for several different routes of administration,including oral exposure to triclosan-containing products (e.g., toothpaste), oral ingestion ofcapsules and aqueous solutions, and percutaneous exposure (in vivo and in vitro). Table 38identifies the studies and their designs based on the route of administration of triclosan.

Table 38: List of Metabolism Studies of Triclosan

Route of Administration Study Design Reference

Oral (capsules) Single versus multiple dose (intra-subjectcomparisons)

Lucker et al., 1990(109)

Treatment versus placebo (inter-subjectcomparisons)

Lin, 1989(135)

Treatment versus placebo (inter-subjectcomparisons); effect of mouth rinse

Colgate-Palmolive, 1988(136)

Oral (toothpaste)

Toothpaste versus toothpaste plus percutaneous(inter-subject comparisons)

BIBRA International, 1988(137);Beiswanger and Tuohy, 1990(138)

Oral (mouth rinse) Treatment versus placebo (inter-subject Lin, 2000



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Route of Administration Study Design Reference

comparisons) (118)

Treatment versus placebo (intra-subjectcomparisons) (soap bar use)

Wagner and Leshar, 1977(139)

Percutaneous (in vivo)

Low versus high dose (hand washing study) Ciba-Geigy, 1973b(120)

Percutaneous (in vitro) Human versus rat Moss et al ., 2000(21)

Biotransformation of triclosan

In general, the studies outlined in Table 38 indicate that, due to a pronounced first-passeffect, only trace amounts of the parent compound (i.e., unconjugated or free triclosan) aredetected in the plasma. As a result of the first-pass effect, there is near total conversion oftriclosan to glucuronide and sulphate conjugates [Lin, 1989 (135) and 2000 (118)].

Studies indicate that the relative amounts of glucuronide- and sulphate-conjugated triclosanpresent in the plasma vary depending on the plasma steady state concentrations of totaltriclosan. Up to study day 14, the main metabolite in plasma was the glucuronide conjugate(97% of total triclosan) in patients receiving 15 mg/day with only trace amounts of sulphate[Lucker et al ., 1990 (109)]. Similarly, the glucuronide conjugate predominated in plasmasamples from subjects brushing daily with triclosan-containing toothpaste (0.6%) [Colgate-Palmolive, 1988 (136)]. However, as steady state concentrations were reached (Cmax =330 ng/mL) during a 36-day exposure to the capsules, the absolute amount of glucuronideconjugates remained relatively constant, whereas sulphate conjugates increased toapproximately 53% [Lucker et al., 1990 (109)].

Two studies comparing oral exposure to triclosan-containing toothpaste alone and incombination with percutaneously applied triclosan-containing products (e.g., soap, talc,anti-perspirant/underarm deodorant) revealed that, in each study and for both groups,circulating metabolites were composed primarily of glucuronide for the duration of the study(8 and 13 weeks) [BIBRA International, 1988 (137); Beiswanger and Tuohy, 1990 (138)].Plasma concentrations of total triclosan generally did not exceed 100 ng/mL in the 8-weekstudy and ranged from approximately 20 to 30 ng/mL in the 13-week study. In the 13-weekstudy [Beiswanger and Tuohy, 1990 (138)], the group receiving oral plus percutaneousexposure had increased blood levels of total triclosan and glucuronide-conjugated triclosan(approximately 24 to 30 and 26 to 29 ng/mL, respectively) compared with the groupreceiving oral exposure only (approximately 19 to 22 and 19 to 21 ng/mL, respectively)throughout the study (Weeks 3, 6, and 13).

In summary, the relative ratio of glucuronide to sulphate conjugates alters with repeat

dosing (i.e., as plasma steady-state triclosan levels are reached) versus single dosing.Generally, lower plasma concentrations of total triclosan are associated with glucuronideconjugates as the predominant metabolite. With increasing plasma levels of total triclosan,there is an increase in circulating sulphate conjugates, which can reach levels greater thanthose attained by glucuronide conjugates, given high enough plasma levels of totaltriclosan.

Metabolism Following Percutaneous Administration

Glucuronidation and sulphation were demonstrated to occur in an in vitro diffusion skinmodel used to assess the ability of skin to metabolise triclosan [Moss et al ., 2000 (21)]. Ofthe radioactivity in the receptor fluid (i.e., following absorption through the skin) at 24

hours, 3.5% of the applied dose was present as triclosan glucuronide and 0.2% was presentas triclosan sulfate.



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A study of percutaneous absorption from 21 days of hand washing with triclosan-containingsoap or scrub showed that triclosan was nearly completely converted to a conjugated formdetectable in plasma (no free triclosan was detected) [Ciba-Geigy, 1973b (120)]. In vivo percutaneous absorption studies have measured plasma concentrations of free plusconjugated triclosan as well as free triclosan following single and multiple applications of

triclosan-containing products (e.g., soap, anti-perspirant, skin cleanser, hand scrub)[Thompson et al ., 1975a (127); Thompson, 1975c (122); Thompson et al ., 1976 (129);Hong et al ., 1976 (24)] (see Table 36). In these studies, although the specific conjugateswere not differentiated (i.e., glucuronic acid and sulphuric acid conjugates), results showedthat the majority of circulating triclosan was in the conjugated form. In a small study, 6subjects used triclosan-containing soap for 45 days, with plasma steady-stateconcentrations ranging from 100 to 2,580 ng/mL. Plasma from subjects with lower steady-state levels contained primarily glucuronide conjugates, whereas plasma from subjects withhigher steady-state levels contained primarily sulphate conjugates [Wagner and Leshar,1977 (139)].

3.3.11.2.4 Excretion

The routes of excretion of triclosan were measured following single and multiple oral doses(triclosan capsules or mouthwash solution), single and multiple percutaneous applications(soap), and intravenous administration. Elimination half-life values for triclosan wereoutlined in the pharmacokinetic section (see Table 35) and are discussed in more detailbelow.

Elimination half-life

The elimination half-life values for orally-administered triclosan are comparable from studyto study, irrespective of the formulations used, the duration of exposure, and the age of thesubjects. For adults receiving single and multiple administrations of triclosan capsules, the

elimination half-life values are 13.42 and 18.97 hours, respectively [Lucker et al., 1990(109)]. In adults, single doses from aqueous solutions and dental slurries have eliminationhalf-life values approximating 11 to 20 hours [Concordia Research Laboratories, 1997a(110); Sandborgh-Englund et al., 2006 (116)], and single use of triclosan-containingtoothpaste has a value of 14.6 hours [Colgate-Palmolive, 1997b (115)]. In children, singledoses of aqueous solution result in elimination half-lives up to 16.8 hours [Colgate-Palmolive, 1997a (111); Concordia Research Laboratories, 1997b (112)]. One studyreported an elimination half-life value of approximately 10 hours based on excretion datafollowing intravenous administration of triclosan [Maibach, 1969 (123)]. This valuesuggests a slightly shorter elimination half-life for intravenously injected triclosan comparedwith values obtained from oral studies, which may be reflecting the fact that absorption isby-passed following intravenous administration.

The elimination half-life value for dermally-applied triclosan used at 1% in a hand washformulation was 1.4 days, based on combined data from men and women [Ciba SpecialtyChemicals, 2002 (134)]. In vivo percutaneous absorption studies for triclosan conducted inthe early 1970s had suggested that differences exist in the rate of elimination betweenCaucasians and African Americans [Thompson, 1975c (122); Thompson et al ., 1976 (129)].In an 8-day study conducted with triclosan-containing hand scrub, the elimination half-livesranged from 33.6 to 50.4 hours for Caucasians and from 271.2 to 374.4 hours for AfricanAmericans [Thompson, 1975c (122)]. In a similar 31-day study, this difference in rate ofelimination was observed between some but not all the African American and Caucasiansubjects [Thompson et al ., 1976 (129)]. Despite these findings, a subsequent study wasdesigned specifically to evaluate any race differences (Caucasians vs. African Americans vs. Asians) in the metabolism of triclosan [Beiswanger and Tuohy, 1990 (138)]. This study was

conducted with triclosan-containing toothpaste, soap and deodorant, and revealed nometabolic differences between these populations.



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Elimination half-life

The elimination half-life values for orally-administered triclosan are comparable from studyto study, irrespective of the formulations used, the duration of exposure, and the age of thesubjects. For adults receiving single and multiple administrations of triclosan capsules, the

elimination half-life values are 13.42 and 18.97 hours, respectively [Lucker et al., 1990(109)]. In adults, single doses from aqueous solutions and dental slurries have eliminationhalf-life values approximating 11 to 20 hours [Concordia Research Laboratories, 1997a(110); Sandborgh-Englund et al., 2006 (116)], and single use of triclosan-containingtoothpaste has a value of 14.6 hours [Colgate-Palmolive, 1997b (115)]. In children, singledoses of aqueous solution result in elimination half-lives up to 16.8 hours [Colgate-Palmolive, 1997a (111); Concordia Research Laboratories, 1997b (112)]. One studyreported an elimination half-life value of approximately 10 hours based on excretion datafollowing intravenous administration of triclosan [Maibach, 1969 (123)]. This valuesuggests a slightly shorter elimination half-life for intravenously injected triclosan comparedwith values obtained from oral studies, which may be reflecting the fact that absorption isby-passed following intravenous administration.

The elimination half-life value for dermally-applied triclosan used at 1% in a hand washformulation was 1.4 days, based on combined data from men and women [Ciba SpecialtyChemicals, 2002 (134)]. In vivo percutaneous absorption studies for triclosan conducted inthe early 1970s had suggested that differences exist in the rate of elimination betweenCaucasians and African Americans [Thompson, 1975c (122); Thompson et al ., 1976 (129)].In an 8-day study conducted with triclosan-containing hand scrub, the elimination half-livesranged from 33.6 to 50.4 hours for Caucasians and from 271.2 to 374.4 hours for AfricanAmericans [Thompson, 1975c (122)]. In a similar 31-day study, this difference in rate ofelimination was observed between some but not all the African American and Caucasiansubjects [Thompson et al ., 1976 (129)]. Despite these findings, a subsequent study wasdesigned specifically to evaluate any race differences (Caucasians vs. African Americans vs.

Asians) in the metabolism of triclosan [Beiswanger and Tuohy, 1990 (138)]. This study wasconducted with triclosan-containing toothpaste, soap and deodorant, and revealed nometabolic differences between these populations.

Excretion Following Oral Administration

Following both single and multiple oral doses of triclosan in a gelatine capsule, thepredominant route of excretion is the urine (57 to 87% of the administered dose), withmuch smaller amounts appearing in the faeces (10 to 33% of the administered dose)[Lucker et al., 1990 (109); Stierlin, 1972b (121); Ciba-Geigy, 1976c (140)]. In a studyusing single doses of aqueous solutions containing triclosan, the major fraction wasexcreted within 24 hours of exposure, with between 24 and 83% (median 54%) of the oraldose excreted within the first 4 days after dosing [Sandborgh-Englund et al., 2006 (116)].In the same study, the median urinary excretion half-life was 11 hours and relative renalclearance was 57% of the total dose. Following single oral administration of radioactivetriclosan (in capsule form), nearly 100% of the radioactivity detected in the urine wasreported to be the glucuronide conjugate [Stierlin, 1972b (121); Ciba-Geigy, 1976c (140)],whereas in the faeces, 30 to 40% was recovered as the free unchanged triclosan compound[Stierlin, 1972b (121)].

Excretion Following Percutaneous Administration

The predominant route of excretion following percutaneous application of triclosan is theurine (2 to 14% of the applied dose) [Stierlin, 1972b (121); Caudal et al., 1974 (125);Thompson et al ., 1975b (128)], with much smaller amounts appearing in the faeces (0.5 to

2% of the applied dose) [Stierlin, 1972b (121)]. Excretion data revealed that, followingsingle and multiple applications (2 to 3 times/day for 45 days), all the triclosan appearing in



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the urine was present as the glucuronide conjugate [Wagner and Leshar, 1977 (139);Caudal et al., 1974 (125)], whereas the majority of the faecal triclosan was present as thefree unchanged compound [Wagner and Leshar, 1977 (139)].

Excretion Following Intravenous Administration

Excretion data obtained from an intravenous study were consistent with those obtainedfrom the oral studies. Following injection of radiolabelled triclosan, the majority of the dose(approximately 65%) was excreted in the urine, while approximately 21% was excreted inthe faeces [Maibach, 1969 (123)], suggesting biliary excretion into the gut.

3.3.11.2.5 Plasma Protein Binding

An in vitro study was conducted with serum derived from a healthy donor’s blood toestimate the binding of 14C-labelled GP 41353 to serum proteins (specifically albumin) usingequilibrium dialysis. At concentrations ranging from 0.36 to 7.9 µg/mL, 99.8% of the drugwas bound to serum proteins. There was no indication of saturation of binding sites, and theratio of the unbound fraction to the bound fraction remained constant with increasing

concentrations. At concentrations ranging from 0.095 to 9.1 µg/mL, 98.9% of the drug wasbound to albumin, with no indication of saturation of binding sites. It was thereforeconcluded that, in plasma, the majority of the drug binds to albumin. Equilibrium dialysisshowed that dialysis time is 60 minutes and that GP 41353 is bound to protein more firmlythan it adheres to the membrane [Wagner, 1973 (141)].

3.3.11.2.6 Exposure of Infants by breast milk

Distribution into Breast Milk

Infant exposure to triclosan was estimated based on levels of triclosan found in 5 randomsamples of breast milk in a small study [Adolfsson-Erici et al ., 2002 (102)]. In this study,

the concentrations of triclosan in breast milk were reported to range from less than 20 to300 µg/kg lipid weight. Based on breast milk fat intake rates of 0.0268 kg breast milkfat/day and using the most conservative (highest) concentration for intake estimates, theexposure of infants to triclosan via breast milk was calculated to be approximately 8.04µg/day (0.00804 mg/day). Assuming a small infant of 2 kg weight, the exposure totriclosan from breast milk was calculated to be 4.02 µg/kg bw/day.

Another study determined triclosan levels in plasma and milk from 36 Swedish nursingmothers with and without known exposure from personal care products [Allmyr et al.,2006]. In this study triclosan was found in breast milk at concentrations ranging from <0.018 to 0.95 ng/g (levels comparable on a fresh weight basis for milk to those determinedby Adolfsson-Erici et al.). Allmyr et al. (Ref 103) calculated that the daily intake of triclosanwould be <11–570 ng/day for an infant weighing 4 kg with an estimated milk intake of 150ml/kg/day. As breast milk levels are lower than those in plasma of nursing mothers (ratioM/P <1), the infant is exposed to a considerably smaller dose of triclosan via the breast milkcompared to the dose in the mother.

A recent risk assessment estimated the maximum daily consumption of triclosan by infantsthrough breast milk, based on the most conservative values for breast milk concentration[Dayan, 2007 (104)]. The study based its calculations on the average triclosanconcentration in the 5 breast milk samples with the highest triclosan levels, out of 62samples, as well as a value for infant milk intake covering 97.5% of the population with thehighest intake on a volume/kg body weight basis, i.e., 1-month-old infants. The maximumdaily infant consumption of triclosan through breast milk was calculated to be 7.4 µg/kgbw/day.



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3.3.11.2.7. Triclosan levels in urine and plasma

Information on exposure to triclosan can be derived from biomonitoring and similar studies.Urinary triclosan concentrations were assessed in a representative sample of the U.S.general population from the 2003-2004 National Health and Nutrition Examination Survey(NHANES) by Calafat et al. (2008) [AR2]. They analyzed 2,517 urine samples by means of

sensitive analytical methods and detected triclosan in about three-quarters of urinesamples: The geometric mean and 95th percentile concentrations were 13.0 μg/L (12.7μg/g creatinine) and 459.0 μg/L (363.8 μg/g creatinine), respectively. Concentrationsdiffered by age and socioeconomic status but not by race/ethnicity and sex. Specifically, theconcentrations of triclosan appeared to be highest during the third decade of life and amongpeople with the highest household incomes.Dose estimates for triclosan based on the NHANES measurement data have not beenreported by Calafat et al. since the conversion of measured spot urine concentrations todaily doses is not trivial because of several uncertainties related to variable dilution causedby wide variations in fluid intake and excretion and lack of information on route of exposure(i.e. oral, dermal, inhalation) and duration. Three methods proposed to estimate the dosefrom measured spot urine concentrations in the absence of total urine volume data havebeen recently used by the US EPA in computations for an aggregate risk assessment ontriclosan (US EPA 2008; AR9). SCCP considers these dose estimates for consumers in the US as useful additionalinformation in their evaluation on the safety of triclosan.

There are three smaller studies on plasma levels in consumers, two in Swedish and one inAustralian subjects: Sandborgh-Englund et al. [ref. 116] found triclosan in plasma at 0.1–8.1 ng/ml of 10 subjects, of which 5 were exposed and 5 not exposed to triclosan viapersonal care products. In that study there was no apparent difference between the twogroups. Allmyr et al. 2006 (103) detected a broader range of plasma concentrations(0.010–38 ng/ml) in 36 Swedish nursing mothers, with higher concentrations in bothplasma and milk of individuals with known exposure to triclosan via personal care products.Recently, Allmyr et al. 2008 [AR1] reported on their analysis of human blood samples

collected in Australia between 2002 and 2005, and pooled according to age, gender andregion. The dataset suggests that the exposure to triclosan among different groups of theAustralian population is relatively homogeneous [Allmyr et al. 2008]. In comparison to theirprevious measurements in human plasma from Sweden, triclosan concentrations were afactor of 2 higher in Australian serum than in Swedish plasma.

3.3.11.2.8 Summary of Human Pharmacokinetic Data

Triclosan is very well absorbed following oral ingestion (up to 98% of the dose). However,under normal conditions of toothpaste use (i.e., expectoration and rinsing) or followingpercutaneous application of several different personal care products, there is only limitedabsorption (approximately 5 to 10% of the dose via either of these routes ofadministration). Based on plasma levels and percentage of dose absorbed, it is clear thatlow exposures to triclosan occur following either toothpaste use or soap/hand wash use andthat, with repeated exposures using either route, low steady-state levels of triclosan arereached after approximately 7 to 10 days.

Regardless of the formulation administered, only trace amounts of the parent compound aredetected in the plasma following exposure to triclosan-containing products. Due to apronounced first-pass effect, there is near total conversion of absorbed triclosan toglucuronic and sulphuric acid conjugates. The relative proportions of these metabolites varydepending on the plasma steady state concentration of total triclosan, with higherconcentrations resulting in a shift from predominantly glucuronide- to predominantlysulphate-conjugates. Following ingestion, percutaneous application, or intravenous

administration of triclosan, the predominant route of excretion is the urine, in which



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triclosan is present as the glucuronide conjugate. In contrast, triclosan excreted in thefaeces is present as the free unchanged compound.

Pharmacokinetic data, in particular, AUC values after single or repeated oral exposures(e.g., after toothpaste use), as well as plasma levels after dermal (soap application)exposures, indicate a lack of evidence of bioaccumulation of triclosan.

3.3.11.3 Human Irritation and Sensitisation

Several irritation, sensitisation, and photosensitisation studies have been conducted withtriclosan in humans. Data regarding the irritation and/or sensitisation potential of triclosanwas also available from studies that reported results of routine patch tests with a series ofcompounds in patients presenting with contact dermatitis.

3.3.11.3.1 Irritation/Corrosivity

The irritation potential of triclosan was determined in tests on human skin and human

mucous membranes. The main findings of these studies are presented in Table 39.

Table 39: Findings from Irritation Studies with Triclosan in Humans

No. ofsubjects

Application Details Major Findings Reference

N=106females.

Repeated Insult Patch Test.Soap formulation applied to theback for 48 hours, twice a weekfor 5 weeks. Concentration oftriclosan not reported (testmaterial described as “soap bar”and “P-300 Anti-Bac. Deo. Soap(NDA #16-486)”)

There was no evidence of primaryirritation following any of the 48-h patchtests (a total of 1155 patch tests).


N=20volunteers

Hand washing Study. Volunteerswith no known allergic reactionsto hand washing agents. Acommercially available 2%triclosan detergent was used(commercially available).Subjects washed with 5 mL for 1min, rinsed, and applied afurther 5 mL for 2 min ofwashing before rinsing anddrying thoroughly. Thisprocedure was performed 5times at 20 min intervals.

Five of 20 subjects (25%) complained ofirritation consisting of itching andsoreness. In all cases, the irritationreaction was delayed by 6 to 8 h afterthe use of the triclosan-containingdetergent. In 4/5 cases, the reactionlasted 24 to 36 h. In the worst case, thecomplaint was of a burning sensation andslight swelling that persisted for 2 days.It is important to note that this study didnot use a control group treated with adetergent formulation without triclosanand it was inconclusive whether theirritation reaction was due to triclosan or

to some other component of thedetergent formulation.

Bendig, 1990(162)

N=10subjects

Finn Chamber Patch test. Testswere conducted on the forearmsof subjects. Single application ofpatches saturated with either1.0% SLS, 0.3% triclosan, 1.0%SLS and 0.3% triclosan, ordistilled water.

No skin reactions were observed withtriclosan alone or distilled water. SLSresulted in erythema of the skin of allsubjects. SLS and triclosan together didnot result in erythema. Pre-treatmentwith triclosan did not reduce erythemaresulting from exposure to SLS.

Barkvoll andRolla, 1994(143)

N=19subjects

Occluded patch test, double-blind study. 1 cm of each of 4toothpastes was applied to theforearm of each subject for 24hours. Concentration ofingredients in toothpastes wasnot reported.

One triclosan-containing toothpaste(triclosan/copolymer/SLS/ propyleneglycol) produced mild to severe irritantreactions in 16 of 19 subjects, while theother (triclosan/zinc citrate/SLS/polyethylene glycol) did not elicit anyreaction.

Skaare et al .,1997a(144)



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No. ofsubjects


N=14 or 15subjects

Five mouth rinses were tested ingroups of subjects: A, 1.5%sodium lauryl SLS B, 1.5%SLS/0.5% zinc citrate; C, 1.5%SLS/polyethylene glycol (1:8);D, 1.5% SLS/0.15%triclosan/0.3% zinc citrate; E,1.5% SLS/0.15% triclosan

Mouth rinses containing triclosan (D, E)resulted in decreased frequency andseverity of erythemic reactions,compared to mouth rinses withouttriclosan (A, B, C). Mouth rinse D elicitedonly 2 erythema reactions and E elicited5 reactions. These 5 subjects also hadmild desquamation.

Skaare et al .,1997b (145)

Experiment I:N=10subjects

ExperimentII: N=28subjects

Irritation tests in oral mucosa(daily exposures). Experiment I(5 min/d for 5 d): Solidox Fluor (F) (1.5% SLS) or Solidox G (G) (1.5% SLS/0.3%triclosan/0.75% zinc citrate).Experiment II (2 min twice dailyfor 4 d): A, 1.5% SLS; B, 3%SLS/0.3% triclosan/ 0.75% zinccitrate; C, detergent-freetoothpaste (negative control).

None of the subjects using Solidox G (triclosan-containing toothpaste) showedoral mucosal desquamation, versus 7/10subjects with positive results usingtoothpaste without triclosan.Comparison of results overall showedthat triclosan eliminated the effects of1.5% SLS (Experiment I) and reducedthe severity of the effects of 3% SLS(Experiment II).

Skaare et al., 1996 (146)

Abbreviations: SLS = sodium lauryl sulfate

The ability of triclosan to cause irritation to human skin or mucous membranes wasevaluated in human volunteers. Triclosan (0.3%) was shown not to induce skin irritation insingle patch tests in 10 subjects [Barkvoll and Rolla, 1994 (143)]. There was also noevidence of primary skin irritation in repeated patch tests with a bar soap formulationcontaining triclosan (concentration of triclosan not specified) in 106 female subjects[Colgate-Palmolive, 1972 (147)]. In this study, a total of 1,155 patch tests wereconducted, including a challenge application 14 days after the 10th patch test in eachsubject. Although irritation effects were observed in a hand washing study using acommercial detergent containing a high concentration (2%) of triclosan, the irritation effectscould not clearly be attributed to triclosan [Bendig, 1990 (162)]. Thus, taken altogether,

the data indicated that triclosan showed low skin irritation potential in clinical irritation testsand, possibly, skin irritation in a commercial detergent formulation containing a highconcentration of triclosan (2%).

The effect of triclosan on skin and oral mucosa irritation produced by SLS was investigatedin four studies. In the same patch test study in which triclosan was shown not to produceskin irritation, it was also shown to eliminate SLS-induced irritation [Barkvoll and Rolla,1994 (143)]. In another study, the effects of triclosan in toothpaste formulations on SLS-induced irritation was tested in skin, with inconclusive, but suggestive, results with respectto a protective effect of triclosan [Skaare et al ., 1997a (144)]. Studies of oral mucosalresponse to various mouth rinse or toothpaste formulations containing SLS (1.5 or 3%)with/without triclosan (0.15% or 0.3%) showed that formulations containing triclosan

reduced or eliminated the severity and frequency of SLS-induced oral mucosal erythemaand desquamation compared to formulations without triclosan [Skaare et al ., 1997b (145);Skaare et al., 1996 (146)]. Taken altogether, the data from these studies indicate thattriclosan has a protective effect against SLS-induced skin or oral mucosa irritation. Whilethe effects of triclosan alone have not been evaluated in stand-alone tests in oral mucosabecause triclosan was typically tested in combination with SLS, the lack of irritant effects oftriclosan-containing test formulations in oral mucosa studies, together with the protectiveeffects of triclosan against SLS-induced irritation, indicate that triclosan is not a mucosalirritant.In summary, the skin and oral mucosa irritation studies evaluating the effects of triclosanalone, or in combination with SLS, indicate that triclosan is not a skin or oral mucosalirritant.



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3.3.11.3.2. Sensitisation

Table 40: Findings from human induction studies

No. of

Subjects


106females

Repeated Insult Patch Test.Induction: Soap formulationapplied to the back for 48hours, twice a week for 5weeks (occluded).Challenge: application 14 dafter 10th application.Concentration of triclosannot reported in P-300 Anti-Bac. Deo. Soap.

There was no evidence of sensitisationpotential in any of the 106 volunteers.

Colgate-Palmolive,1972(147)

144 males Modified Draize test (patchtest). Induction: triclosan inpetrolatum was applied for

48-72 h per application, 10times over 3.5 weeks.Challenge: After 14 d, usinga patch for 72 h.

There was no evidence of sensitisationpotential in any of the tests conducted withinduction/challenge combinations of 5%/5%

(none of 61 subjects tested), 20%/1% (noneof 83 subjects tested).

Marzulli andMaibach, 1973(148)

20males /females

Modified Maximisation Test.Preparation: 5% sodiumlauryl sulphate underocclusion for 24 h.Induction: Application of20% triclosan in petrolatumat the same site on Days 1,3, 5, 7, and 9. Challengewas 14 d later, using 1%,2%, or 5% triclosan.

Induction phase: 17/20 subjects showed signsof skin irritation (erythema, slight oedema,moderately painful). However, 0/20 testvolunteers and all control volunteers showedno positive patch test reactions up to 7 dayspost-challenge.

Lachapelle andTennstedt, 1979(18)

150, sex ofsubjectssex notspecified

Repeat Insult Patch Test(100 volunteers) and theProphetic Patch Test (50volunteers). Applications(0.5 mL) of triclosan insolution or as a slurry werefor 24 h (no further detailswere provided).

There was no evidence of skin sensitisation inany of the 150 volunteers (note that theconcentration of triclosan tested was notprovided).

DeSalva et al .,1989(1)

In total, triclosan was tested in 420 healthy subjects using variations and modifications tothe Patch, Draize, and Maximisation Test methods at induction concentrations of up to 20%and challenge concentrations of up to 5%. There were no positive reactions in any of thetest subjects, including after repeated patch testing [e.g., Colgate-Palmolive, 1972 (14);DeSalva et al ., 1989 (1)]. No positive challenge results were observed, leadinginvestigators to conclude that triclosan has a very low sensitisation potential. Takenaltogether, the results from these studies indicate that triclosan has very low sensitisationpotential in healthy subjects.

CommentSCCP considers human induction studies as unethical

Triclosan has also been tested in patients with contact dermatitis or suspected cosmeticallergy. The results of routine patch testing with triclosan as one of a series of preservativeor antimicrobial ingredients tested in these patients are shown in Table 41. The data showthat triclosan has a low potential to cause positive skin reactions in this sensitive population.

Table 41: Findings from Patch Testing with Triclosan in Patients with, or Suspected ofHaving, Contact Dermatitis



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No. of PatientsTested

Major Findings Reference

5,202 (cosmeticintolerance)

Patch test. Seven (7) of 5,202 patients (0.1%) had a positivereaction to triclosan. In particular, 1 of 156 patients with a known “pure” cosmetic allergy showed a positive reaction (0.6%). Note:

concentration of triclosan was not reported.

Broeckx et al ., 1987(149)

627 (suspectedcontact dermatitis)

Patch test. No positive reactions to triclosan (2% in petrolatum)were observed (0%).

De Groot et al .,1986(150)

11,406 (consecutivepatients)

Patch test. Twenty-nine (29) of 11,406 patients (0.3%) had apositive reaction to triclosan (2% in petrolatum). Note that 59 of11,406 (0.5%) patients had a questionable/irritative reaction.Investigators considered the sensitisation rate to be low or verylow.

Schnuch et al ., 1998(151)

179 (suspectedcosmetic allergy)

Patch test. Two (2) of 179 patients (1.1%) had a positive reactionto triclosan (2% in petrolatum)

De Groot et al .,1985(152)

3 (patients known to

have used creamcontaining 3%triclosan)

All 3 patients in these case studies tested positive in patch tests

(2% triclosan in petrolatum) at 48, 72, and 96 h.

Veronesi et al ., 1986

(153)

2,295 (suspectedallergic contactdermatitis)

Patch test. Triclosan was described as having a low sensitisationrate, based on the observed rate of 0.8% positive reactions totriclosan (2% in petrolatum).

Perrenoud et al .,1994 (154)

2,002 (consecutivepatients)

Patch test. In total, 0 of 432, 0 of 470, and 2 of 1,100 patientstested positive to 0.5% triclosan in Vaseline, 1.0% triclosan inethanol, and 2.0% triclosan in Vaseline, respectively (i.e., 0%,0%, and 0.18% positive reactions, respectively).

Wahlberg, 1976(155)

103 (suspectedcontact dermatitis)

Patch test. Three (3) of 103 patients (2.9%) tested positive. Ofthe 3 positive reactions, 2 patients were known to have usedcream containing 3% triclosan (the 3rd patient’s history of use oftriclosan was unknown). Note: triclosan concentration tested was

not reported.

Steinkjer andBraathen, 1988(156)

1,796 (eczemapatients withsuspected contactallergy)

Patch test (chamber method). One (1) of 1,796 patients (0.06%)tested positive to 1% triclosan.

Hannuksela et al .,1976(156)

1,234 (consecutivepatients witheczema)

Patch test. There were reported to be 1 to 2% positive reactionsin 1,234 patients tested with 2% triclosan in petrolatum.

Mitchell et al ., 1982(157)

713 (suspectedcosmetic dermatitis)

Patch test. One (1) of 713 patients (0.14%) with suspectedcosmetic dermatitis tested positive to triclosan. Note: triclosanconcentration tested was not reported.

Adams and Maibach,1985(158)

745 (suspected sun-related skin disease)

Photopatch test trial. One (1) of 745 patients (0.13%) testedpositive for a contact allergic reaction to 2% triclosan in

petrolatum; 2 of 745 (0.27%) tested positive for a photoallergicreaction.

Wennersten et al .,1984

(160)

Case reports of contact dermatitis due to triclosan use have been relatively rare (Campbelland Zirwas 2006, AR3), although a few cases have been reported following the use of acream formulation containing a high concentration of triclosan (3%) together with 0.02%flumethasone pivalate. Three such cases were reported by Veronesi et al . [1986 (153)],and 2 by Steinkjer and Braathen [1988 (159)]. The reason for a third case of reportedallergic contact reaction to triclosan was not discovered [Steinkjer and Braathen, 1988(159)]. All 6 of these patients were found to have positive results in patch tests using 2%triclosan in petrolatum (see Table 41). Investigators concluded that low concentrations oftriclosan in cosmetic products do not cause contact dermatitis; however, sensitisation may

occur following the use of products containing higher concentrations [Veronesiet al

., 1986(153)].



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In general, the clinical test results have shown that triclosan has a very low sensitisationpotential. This is apparent in the interpretation of results from extensive testing in patientswith known or suspected allergic contact dermatitis. In total, over 14,000 consecutivepatients have been tested for reaction to triclosan (typically tested at a concentration of 2%in petrolatum), with the range of positive results being 0.1 to 0.3% of the testedpopulation. Additionally, triclosan testing in patients with known or suspected cosmetic

allergy or intolerance has shown positive reaction rates ranging from 0.06 to 0.8% of a totalof 11,887 tests conducted in this population.

3.3.11.3.3 Photo-Induced Toxicity

The potential of triclosan to induce photosensitisation reactions was determined in a numberof studies. The major findings of these studies are presented in Table 42.

Table 42: Findings from Phototoxicity and Photosensitisation Studies with Triclosan inHuman Subjects


N=5 males. 100 µL (triclosan, 0.1% in methanol,0.1 or 1.0% in petrolatum) applied to a 30 cm2 areaon the back. Exposed to light sources 1 hour afterapplication; readings at 24 and 48 h.

There was no evidence of phototoxicity. Urbach, 1973(101)

N=104 females. Subjects had undergone repeatedinsult patch testing (soap formulation withconcentration of triclosan not reported; 10applications and a challenge after 14 d – allnegative). Photochallenge: 24 h after challenge andagain 7 days after 1st photochallenge.

There was no evidence ofphotosensitising potential following thephotochallenge application. After thesecond UV light photochallenge, onesubject had a minimal reaction,considered to be the result ofscratching, which was completelynegative by the 48-h reading.


Phototoxicity (n=10): triclosan (2.5% in petrolatum)applied to skin for 1 hour, followed by irradiation.

Readings at 4-6 and 24 h.Photoallergy (n=25): triclosan (10% in petrolatum)applied to the same site for five 48-hour intervalsunder occlusion. Site was irradiated after eachapplication. A new site was photopatch tested 2weeks after the last 48-h test.

There was no evidence of phototoxicityor photoallergenicity.

Kligman, 1969(161)

Modified Draize test (patch test). Induction:triclosan in petrolatum was applied for 48-72 h perapplication, 10 times over 3.5 weeks. Elicitation:After 14 d, using a patch for 72 h. Three (3)minimal erythema doses (MED) of Kromayer lightwere used during the induction phase, and 10 MEDfiltered through window glass during the elicitationphase.

There was no evidence ofphotosensitisation ininduction/challenge combinations using1%/1%, 5%/1%, 20%/1%, and20%/5% concentrations of triclosan(i.e., 0/51, 0/60, 0/52, and 0/24positive responses in the tests,respectively, for a total of 0/187subjects).

Marzulli andMaibach, 1973(148)

Photopatch test trial. 745 (patients with suspectedsun-related skin disease (photodermatitis)) weretested for reaction to triclosan (2% in petrolatum)as part of a standard series of tests.

2 of 745 (0.27%) tested positive for aphotoallergic reaction.

Wennersten et

al ., 1984(160)

Patch and photopatch test. Triclosan (2% inpetrolatum) was tested; details not provided. 103patients with suspected contact dermatitis.

No positive photoallergic reactions. Steinkjer andBraathen, 1988(159)

Six studies investigated the photosensitising potential of triclosan. In one of the largerstudies using a soap formulation containing triclosan (concentration not reported), only onepositive reaction was observed following a second photochallenge with the soapformulation; however, this reaction was considered to be the result of scratching [Colgate-

Palmolive, 1972 (147)]. There was no evidence of photosensitising potential of triclosan intwo of the other studies that tested over 100 subjects per study, and only two of 745



SCCP/1192/08


patients tested positive for a photoallergic reaction in the largest study (0.27% of patients)[Wennersten et al ., 1984 (160)]. There was also no evidence of phototoxicity orphotoallergenic potential in two smaller studies (n#25) with triclosan at concentrations of upto 10% in petrolatum or 0.1% in methanol.In summary, data from the photosensitisation and patch testing studies performed withtriclosan indicate that it is unlikely to produce phototoxicity or photosensitisation in human

skin at levels used in personal care products. Triclosan was tested at concentrations of upto 10% in petrolatum in the photosensitisation studies.

3.3.12. Special investigations

Special investigations have been conducted to study potential neurotoxic and nephrotoxiceffects of triclosan in rats. In the 14-day neurotoxicity study, clinical signs, organ weights,and brain and nerve histopathology were examined. In the nephrotoxicity study, kidneytissue function from triclosan-treated rats was assessed in vitro. In addition to thesestudies, the effects of triclosan in rodent liver have been evaluated in rats, mice, andhamsters. Studies have been conducted to determine the effect of triclosan on liver

morphology (e.g., size, hepatocyte necrosis, hepatocyte proliferation, changes inhepatocellular organelles) and on biochemical parameters (e.g., protein content,cytochrome P450 content and activity, and fatty acid oxidation activity).

3.3.12.1 Effects of Triclosan in the Brain

A 2-week neurotoxicity study was conducted with triclosan in the rat [Ciba-Geigy, 1973a(163)], the main findings of which are provided in Table 43. No histopathological changeswere observed in the brain or sciatic nerve of treated or control animals. There were nodifferences in brain weights between treated and control animals. Clinical signs includeddecreased movement and muscular tone, polydipsia, and polyuria at dose levels of 300mg/kg body weight/day and higher. The results of this study indicate a NOEL of 100 mg/kgbody weight/day for triclosan in the rat. There was no evidence of neuropathology at anydose level, as examined in the brain and sciatic nerve tissues. The investigators concludedthat triclosan produces no specific neurotoxic effects in the rat; however, the reasons forobservations of clinical signs consistent with possible neurotoxicity (e.g., hypoactivity,decreased muscular tone) are unclear.

Table 43: Findings from a Two-Week Oral Neurotoxicity Study with Triclosan in the Rat

Species

(Strain)

Dosing Regimen

(mg/kg bw/day)

Major Findings Reference,

GLP and OECD

Status

Rat,albino

SIV 50

0, 100, 300,1,000, or 2,000

mg/kg bw/day via oral administration(specific route notreported) for 14days

17 deaths in high-dose group (5 of these were sacrificed dueto moribund condition). Decreased body weights in high-

dose group. Dose-dependent inhibition of movement,decreased muscular tone, polydipsia and polyuria at doselevels of 300 mg/kg bw/day and higher. Clinical signs weremost severe in the high-dose group and also included spasticrespiration for several animals. No difference in brainweights between treated and controls (no other organweights were measured). No histopathological changes inthe brain or sciatic nerve of treated or control animals wereobserved (no other tissues evaluated).NOEL: 100 mg/kg bw/day

Ciba-Geigy,1973a

(163)


3.3.12.2 Effects of Triclosan in Kidney

The nephrotoxic effects of triclosan have been investigated in a non-GLP study (publishedreport) [Chow et al., 1977 (164)], described in Table 44 by means of in vitro and in vivo



SCCP/1192/08


methods. Data from renal cortical slice incubation assays that measured p-aminohippurate(PAH) and N -methylnicotinamide accumulation as a measure of nephrotoxicity indicate thattriclosan may have potential to cause kidney damage, although blood urea nitrogen (BUN)measurements in the same animals did not show indications of overt nephrotoxicity. Thereason for the discrepancy between the in vitro results (where triclosan was added to theincubation medium) and in vivo results (where triclosan was administered to rats) is

unclear, but may be related to the amounts of triclosan that reached the kidney, potentialformation of a metabolite that selectively alters PAH accumulation, or the relativesensitivities of the assays.

Table 44: Findings from a Nephrotoxicity Study for Triclosan

Species

(Strain)

Dosing

Regimen

Duration of

Treatment


GLP and OECD

Status

Rat (Wistar) 625, 1,250,or 1,875mg/kg bw24 h prior to

kidneycortical sliceassay

Single dose In vitro assay: There was decreasedaccumulation of both PAH and NMN. In vivo study: No change in blood urea nitrogenconcentration at 24 h. Time-course study of

renal cortical function in high-dose rats showedinitial decreases in PAH accumulation thatrecovered to near-control levels by 72 h.N -Methylnicotinamide (NMN) accumulation wasnot significantly different from control up to 72h. The significant decreases in PAHaccumulation were dose-dependent.

Chow et al.,1977(164)


In addition to these early toxicity data, a later (1994) GLP study conducted in hamstersshowed that doses of 350 or 900 mg/kg body weight/day in the diet induced renal tubularepithelium proliferation that was evident at 7 and 13 weeks in the 13-week study [SeeTable 45, Persohn, 1994 (167)]. The authors concluded the increased labelling index (LI) inkidney tubular epithelium was a compensatory response to cell damage in the kidney.However, no histopathology results were available for confirmation of the existence of celldamage.

In summary, findings in rat and hamster kidney studies of decreased kidney function and ofincreased cell proliferation that may be secondary to cellular damage suggest that triclosanmay induce kidney damage at relatively high-dose levels.

3.3.12.3 Effects of Triclosan in Rodent Liver

The effects of triclosan on liver morphology, hepatocyte proliferation, and biochemicalparameters, including the activities of cytochrome P450 enzymes, have been investigated ina number of studies in mice, rats, and hamsters. The major findings of these studies are

presented in Tables 45 and 46.3.12.3.1 Cell Proliferation in Rodent Liver

The effect of triclosan on hepatocyte replicative DNA synthesis has been examined instudies conducted in the CD-1 mouse, Sprague-Dawley rat, and Syrian hamster at dosesranging from 25 to 900 mg/kg body weight/day [Eldridge, 1993 (165); Persohn and Molitor,1993 (166); Persohn, 1994 (167)]. In both the mouse and hamster cell replication studies,the effect of triclosan on replicative DNA synthesis was monitored by using the proliferatingcell nuclear antigen (PCNA) technique, the results of which are summarized in Table 45.



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Table 45: Findings from Cell Proliferation Studies for Triclosan

Species(Strain)

DosingRegimen

Duration ofTreatment

Major Findings Reference,GLP and OECD

Status

Mouse(CD-1)

Oral (diet)doses of 0,25, 75, 200,350, or 900mg/kg bw/d

90 days (45day interimdata alsoavailable)

In general, labelling index (PCNA-stained S-phase cells) increased with dose and time, andwas slightly greater in males vs. females. Cellproliferation was significantly increased at 90days by 4.8-, 3.5-, 11-, and 15-fold in M atdoses of 75, 200, 350, and 900 mg/kg/d,respectively, and by 1.5-, 3.3-, 6.1-, and 7.1-fold in F. Control group labelling indices were0.035±0.016 and 0.042±0.036 in males andfemales, respectively. Cell proliferation was notincreased at 25 mg/kg bw/d. Liver morphologychanges showed dose-related hepatocytehypertrophy starting at 25 mg/kg, together withnecrosis of individual hepatocytes. Hypertrophywas reported as the most consistent and

prominent change at 45 d, along with large areasof necrosis in the 350 and 900 mg/kg groups.Necrosis was accompanied by proliferating cells(bile duct epithelial cells, fibroblasts, Kupffercells). Findings at 90 days were comparable tothose at 45 d, except that necrosis wasoccasionally more severe, with panlobularnecrosis in the most severe cases. Mean scoresfor liver necrosis at 90 days were 0, 0, 0.2, 0.6,1.2, and 1.8 for males in the 0, 25, 75, 200,350, and 900 mg/kg groups and 0, 0, 0, 1.2,0.6, and 1.6 for females, respectively (scale of 0-4). Lipofuscin staining, lipid vacuolization, biliaryhyperplasia were minimal.

Eldridge, 1993(165)

GLP and OECDnot specified,but originalanimal studywas GLP-compliant andconsistent withOECD

Rat

(Sprague-Dawley)

Oral (diet)

doses of 0,300, 1,500,or 6,000ppm (actualdoses of 0,~25, ~125,and ~500mg/kgbw/d)

2, 4, 7, 14,

or 42 days(1 groupreceived6,000 ppmfor 14 daysfollowed by28 days ofrecovery)

No deaths occurred, and no clinical signs of

toxicity were observed. Feed consumption wasinitially decreased in high-dose animals, but wassubsequently increased vs. controls. Bodyweight gains were initially decreased, butsubsequently normal in the high-dose group.Absolute and relative liver weights wereincreased at 6,000 ppm compared to controls.Decreased numbers of hepatocyte nuclei permicroscopic field together with increased liverweights indicated hepatic hypertrophy in high-dose animals. Triclosan did not increase cellproliferation at doses of up to 6,000 ppm in thediet, for treatment durations of up to 42 days.Slight but significant decreases in cellproliferation at the high dose were observed

after at least 7 days of treatment. There wereno effects in the lower dose groups. Nohistopathology results were available.

Persohn and

Molitor, 1993(166)

GLP: notspecified

OECD:comparable

Hamster(Syrian)

Oral (diet)doses of 0,75, 200,350, 750, or900 mg/kgbw/d

13 weeks(7-weekdata alsoavailable)

No increases in hepatic labelling indices (LI) vs. control were observed at the high dose of 900mg/kg bw/d at 13 weeks. Kidney tubularepithelial cell nuclear mean LI were examined at200, 350, and 900 mg/kg bw/d doses at 13weeks. Kidney LI was significantly increased inmales at 350 and 900, but not 200 mg/kg bw/d(3.7 and 6X greater than control, respectively).Kidney LI was significantly increased in high dosefemales at 13 weeks (8.8X greater than control).No histopathology results were available.

Persohn, 1994(167)

GLP-compliant.

OECD notspecified, butoriginal animalstudy wasconsistent withOECD



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The cell proliferation data demonstrate that triclosan treatment produces a dose-relatedincrease in cell replication in male and female mice. Morphological examination of liversections from this study revealed various histological changes including hepatocytehypertrophy and necrosis in both sexes of the higher dose groups. In contrast, DNAsynthesis data in male rats, and in male and female hamsters showed that triclosan did notincrease hepatic replicative DNA synthesis in either sex after 7 or 13 weeks of treatment

[Persohn and Molitor, 1993 (166); Persohn, 1994 (167)] (Table 45). Indeed, a significantdecrease in hepatocyte proliferation relative to control was observed in male rats treatedwith ~500 mg/kg body weight/day for at least 7 days, and in male hamsters treated with900 mg/kg body weight/day for 7 weeks. Altogether, the data show that cell proliferationfollowing administration of triclosan is observed in mice, but not in rats or hamsters.

3.3.12.3.2 Liver Morphology and Biochemical Studies for Triclosan

Several investigations into changes in liver morphology and selected biochemicalparameters induced by triclosan have been conducted. Of the six studies, the findings ofwhich are presented in Table 46, three [Molitor et al ., 1992 (168); Molitor and Persohn,1993 (169); and Thomas, 1994 (170)] can be considered to be “pivotal” in the scope of the

endpoints examined within the investigation, even though 2 of the 3 did not containstatements of GLP compliance.

Table 46: Findings from Liver Morphology and Biochemical Studies for Triclosan

Species

(Strain)

Dosing

Regimen

Duration

of

Treatment


GLP and OECD

Status

Mouse(CD-1)

Oral (diet) dosesof 0, 18, 54,258, or 951mg/kg bw/d(males) or 0, 20,

271, or 1,105mg/kg bw/d(females)

14 days +recovery(28 days)

Increases in protein and cytochrome P450(P450) content, and enzyme activities weredose-dependent and reversible. Highlights ofthe results include (high-dose data reported as% of control): increased lauric acid

hydroxylation (up to 833%), ethoxyresorufin O-deethylase activity (up to 502%), andtestosterone hydroxylation (up to 619%);peroxisomal fatty acid beta-oxidation (~340%in M and F) and pentoxyresorufin O-depentylase(up to 2,390 and 1,580% in males and females,respectively). Immunoblot analyses showedincreases in CYP3A1/2 proteins (842 and5,851% in M and F, respectively) and in CYP4Aproteins (~800% in M and F). Electronmicroscopy results show dose-dependent andreversible effects of an increase in smoothendoplasmic reticulum (ER) membranes,reduction and disorganization of rough ERmembranes, and increase in lipid vacuolizationin hepatocytes. Dose-dependent increases innumbers of peroxisomes (“moderate“ to “striking” increases in numbers) at doses of≥54 mg/kg bw/d.

Molitor et al .,1992 (168)

GLP: notspecified

OECD: notapplicable



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Species(Strain)

DosingRegimen

Durationof

Treatment


Status

Rat(Sprague-Dawley)

Oral (diet) dosesof 0, 23, 108, or518 mg/kg bw/d

14 days +recovery(28 days);42 days

Increased, but reversible, absolute and relativeliver weights in high-dose group. Increases inenzyme activities and cytochrome P450 contentwere generally dose-dependent and reversible.Highlights of the results include (high-dose datareported as % control): lauric acidhydroxylation (up to 281%); pentoxyresorufinO-depentylase activity (up to 1,143%).Peroxisomal fatty acid beta-oxidation (FAO) wasunaffected. Immunoblot analyses showeddose-dependent, large, and reversible increasesin CYP2B1/2 enzymes (at least >100X controlat 6,000 ppm); only slight or small increases inCYP3A1/2 and CYP4A proteins (209% and170%of control, respectively). Electron microscopyfindings show reversible proliferation of smoothendoplasmic reticulum, increased cytoplasmiclipid vacuoles, dilated mitochondrial cristae,transient formation of matrical plates in

peroxisomal matrices in high-dose rats.

Molitor andPersohn, 1993(169)

GLP: notspecified

OECD: notapplicable

Hamster(Syrian)

Oral (diet) dosesof 0, 50, 310, or800 mg/kg bw/din males, or 0,46, 314, or 960mg/kg bw/d infemales

14 days +recovery(28 days)

Increased, but reversible, absolute and relativeliver weights in high-dose females. Increases inenzyme activities and cytochrome P450 contentwere generally dose-dependent and reversible.Highlights of the results showed increases in:cytochrome P450 content; ethoxyresorufinO-deethylase and pentoxyresorufin O-depentylase activities; 7α-hydroxytestosteroneproduction in females; 15 α-HT, 16β-HT, andandrostenedione production, lauric acidhydroxylation. Peroxisomal fatty acid beta-oxidation was unchanged. Immunoblotanalyses showed no increases in levels ofCYP1A- and CYP3A-related proteins, but slightto mild increases in CYP4A-related proteins(levels in males were 140% greater thancontrols, but increases in females were slight,with no overall change in total CYP4A levels).Electron microscopy findings show no changesto hepatocytes, including the number and sizeof peroxisomes.

Thomas, 1994(170)

GLP: compliant

OECD: notapplicable

Mouse(ddY); Rat(Wistar)

Daily i.p. dosesof 50 or 100mg/kg bw/d

3 days Triclosan induced aminopyrine N -demethylase(APND) activity slightly, but significantly, inmice at a dose of 100 mg/kg bw/d. Slight(<100% increase vs. control), but significantincreases in biphenyl 4-hydroxylase, APND, andphenacetine O-deethylase activities at 100mg/kg bw/d in rats. Triclosan induced

moderate increases (100-130%) in biphenyl 2-hydroxylase, ethoxycoumarin O-deethylase,and p-nitrophenerole O-deethylase ( p-NPOD)activities at 100 mg/kg bw/d in rats. p-NPODactivity was also increased slightly at 50 mg/kgbw/d in rats.

Kanetoshi etal., 1992 (4)

GLP: notreported

OECD: no

comparableguidelines



SCCP/1192/08


Species(Strain)

DosingRegimen

Durationof

Treatment


Status

Rat(Wistar)

In vitro concentrationsof 0.1, 1, 10, or100 µM

Male ratswere pre-treatedwithcytochromeP450inducersprior topreparationofmicrosomes

Ethoxyresorufin O-deethylase (EROD) andpentoxyresorufin O-depentylase (PROD)activities were inhibited by 93 and 86%,respectively at a concentration of 10 µMtriclosan. Inhibition of phenacetine O-deethylase and 4-nitrophenol hydroxylaseactivities was 5-60% vs. control levels at 10 µMand up to 80% vs. controls at 100 µM.Testosterone 6β-hydroxylase and lauric acidhydroxylase activities were inhibited by up to50% at 100 µM. Ki values for EROD and PRODactivities were 0.24 and 1.48 µM, respectively.The effect on EROD activity was consistent withcompetitive inhibition, whereas the effect onPROD indicated noncompetitive inhibition.

Hanioka et al .,1996 (171)

GLP: notspecified

OECD: notapplicable

Rat(Wistar)

0, 58, 116, or232 mg/kg bw/d

5 days Increases in biochemical parameters weregenerally dose-related, reaching significance atthe high dose in rats. Increases included:

microsomal protein, cytochromes P450 and b5,and NADPH-cytochrome C reductase.Benzyloxyresorufin O-debenzylase (BROD) andpentoxyresorufin O-depentylase (PROD)activities were induced up to 22- and 20-fold,respectively. Levels of other P450 enzymeswere either 2.4- to 4.9-fold increased at thehigh dose, or were unchanged. Immunoblottingdata show levels of CYP 2B proteins (associatedwith PROD activity) were increased from 10.8up to 34-fold.

Hanioka et al .,1997 (172)

GLP: notspecified

OECD: notapplicable

A series of 3 critical studies were conducted in mice, rats (males only), and hamsters toexamine the effects of triclosan on selected biochemical and morphological liver parameters

following dietary administration of triclosan for 14 days [Molitor et al ., 1992 (168); Molitorand Persohn, 1993 (169); Thomas, 1994 (170)]. Biochemical investigations includedmeasurements of levels of protein and cytochrome P450 (P450) content and P450 enzymeactivities. Morphology was examined using electron microscopy (EM).

As can be seen from Table 46, and summarized in Table 47, there exist differences inresponses between species. There was little difference between sexes, although femalesappeared slightly less sensitive to the effects of triclosan than males. With regard tomorphology changes, the most notable was a dose-dependent, “moderate” to “striking”increase in the numbers of peroxisomes in mouse liver electron micrographs [descriptorstaken from the original report by Molitor et al., 1992 (168)]. In contrast, there were noincreases in numbers of peroxisomes in rats and hamsters. Liver weight and microsomalP450 content were strongly increased in mice, mildly increased in higher dose rats and

relatively unaffected in hamsters. Similarly, induction of various enzyme activities wasmost pronounced and occurred at lower doses in mice when compared to other species.Cyanide-insensitive fatty acid β-oxidation activity (palmitoyl-CoA oxidation), a diagnosticindicator of peroxisome proliferation, showed a dose-dependent increase in mice, but not inrats or hamsters. Cytosolic glutathione S-transferase activity was mildly increased in mice,less so in rats, and slightly depressed in hamsters. Lauric acid 12-hydroxylation activity, ascatalysed by isoenzymes of the cytochrome P450 CYP4A gene subfamily, was stronglyincreased in mice but less so in rats and hamsters. Lauric acid 11-hydroxylation activity, ascatalysed by isoenzymes of the cytochrome P450 CYP1A, CYP2B, and CYP2C families, wasagain strongly increased in mice, but less so in hamsters. No effects were found in rats.Ethoxyresorufin O-deethylase (EROD) activity showed a mild dose-dependent increase inmice, with lesser increases in hamsters, and was decreased in rats. A strong induction of

7-pentoxyresorufin O-depentylase (PROD), a CYP2B marker, was found in mice and rats;hamsters showed significantly less potent induction of this activity.



SCCP/1192/08


The pattern of regio- and stereo-selective testosterone hydroxylation was used as a tool toassess treatment-related effects on the activities of several isoenzymes of the microsomalP450 enzyme family. Only in the mouse was total testosterone hydroxylation significantlyinduced. At the highest dose level of 951 mg/kg body weight/day, a 6-fold dose-dependentincrease was observed. The most prominent changes in the testosterone hydroxylationprofile were increased production of the 2β-, 6β-, 15β-, and 16β-hydroxy metabolites.

Production of the 2β- and 6β-hydroxy metabolites is associated with the expression ofcytochrome CYP3A, while 16β-hydroxylation is catalysed by the CYP2B cytochrome family.Rats showed a different pattern of testosterone hydroxylation after triclosan administration.Production of the 16β-hydroxy metabolite was most prominent and, except for a mildinduction of 15α-hydroxylation which is associated with cytochromes CYP2C12 andCYP2C13, no other hydroxy metabolite was produced in significant amounts. Hamsterswere much less sensitive to P450 enzyme induction than mice. The only significant changeobserved in the hydroxylation profile was a small increase in 17-oxidation to formandrostenedione.Immunoblot analyses using monoclonal antibodies generated against, and specific for,inducible isoenzymes of rat liver cytochrome P450 were performed to further clarify thenature of triclosan-induced changes in liver enzymes. Mice demonstrated a slight dose-

dependent decrease in the content of the isoenzymes of the CYP1A gene subfamily. Ratshad an increased content of this isoenzyme family and hamsters a marginal increase.Triclosan strongly induced isoenzymes of the CYP3A subfamily in mice (8.4x control levels),produced a small increase (2X) at the highest dose in rats, and produced a decrease of thisenzyme in female hamsters, with no effect in male hamsters. CYP4A induction was alsoextremely strong (7.8x at the highest dose) in mice and much less so in the rat andhamster (1.6x and 2.4x at the highest dose, respectively).

The effect of triclosan on P450 enzyme activities was also investigated in 3 publishedreports of an in vivo study in mice and in vitro and in vivo studies in rats [Kanetoshi et al., 1992 (5); Hanioka et al ., 1996 (171); Hanioka et al ., 1997 (172)]. Both of the rat studiessupport the findings in the critical 1993 rat investigation [Molitor and Persohn, 1993 (169)].In the in vitro study, the effects of triclosan on P450 enzyme activities were investigated

using liver microsomes from rats pretreated with one of several P450 inducers (3-methylcholanthrene, phenobarbital, pyridine, dexamethasone, or clofibrate) [Hanioka et al .,1996 (171)]. The data from this study, which assessed the effect of triclosan on the activitylevels of P450 enzymes previously increased by P450 inducers, suggest that triclosancompetitively inhibits enzymes of the cytochrome P450 1A family, as indicated by inhibitionof EROD and phenacetin O-deethylase activities, and non-competitively inhibits cytochromeP450 2B enzymes, as evidenced by its effect on PROD activity. Interaction of triclosan withcytochrome P450 2B1/2 enzymes was also suggested by the pattern of induction of variousP450 enzyme activities in the published in vivo rat study which investigated the effects of 5days of dosing with triclosan on P450 enzyme activities [Hanioka et al ., 1997 (172)]. Datafrom the in vivo study show slight to no effects of triclosan administered for 3 days in miceon a variety of P450 activities, but did show increases in P450 activities in rats [Kanetoshi et

al., 1992 (5)]. The reason for the inconsistency with the pivotal 1992 mouse biochemicalinvestigation is unclear, but may be related to the length of time of dosing or the differencein the route of administration (intraperitoneal in this published report vs. dietaryadministration in the pivotal report). Table 47 summarizes the effects of triclosan onselected biochemical parameters.



S C C P / 1 1 9 2 / 0 8

O p

i n i o n o n

t r i c l o s a n

1 1 3

T a b l e

4 7 :

S u m m a r y o f E f f e c t s o n S e l e c

t e d B i o c h e m i c a l P a r a m e t e r s F o l l o w i n g 1 4 - D a y D i e t a r y A d m i n i s t r a t i o n o f T r i c l o s a n

M a

l e R a

t 1

M a

l e M o u s e

2

F e m a

l e M o u s e

2

M a

l e H a m s

t e r

3

F e m

a l e H a m s

t e r

3

E n

d - P o

i n t

E f f e c

t

M i n i m u m

E f f e c

t L e v e

l

( m g

/ k g

b w

/ d a y

)

E f f e c

t

M i n i m

u m

E f f e c t

L e v e

l

( m g /

k g

b w

/ d

a y

)

E f f e c

t

M i n i m u m

E f f e c

t L e v e

l

( m g

/ k g

b w

/ d a y

)

E f f e c

t

M i n i m u m

E f f e c

t L e v e

l

( m g

/ k g

b w

/ d a y

)

E f f e c t

M i n i m u m

E f f e c

t L e v e

l

( m g

/ k g

b w

/ d a y

)

L i v e r w e i g h t ( a b s o l u t e )

↑

5 1 8

↑

5 0

↑

2 7 0

n o n e

↓

9 6 0

P r o t e i n c o n t e n t , m i c r o s o m a l

n o n e

↑

2 0

↑

2 7 0

↑

8 0 0

↑

9 6 0

C y t o c h r o m e P 4 5 0 c o n t e n t

↑

5 1 8

↑

5 0

N D

↑

3 1 0

↑

3 1 4

G l u t a t

h i o n e S - t r a n s f e r a s e

↑

5 1 8

↑

2 5 0

N D

↓

8 0 0

↓ ( s l . )

9 6 0

F a t t y a c i d β - o x i d a t i o n

n o n e

↑

5 0

↑

2 7 0

n o n e

n o n e

L a u r i c

a c i d 1 1 - h y d r o x y l a t i o n

n o n e

5 1 8

↑

2 0

N D

↑

8 0 0

↑

9 6 0

L a u r i c

a c i d 1 2 - h y d r o x y l a t i o n

↑

5 1 8

↑

5 0

N D

↑

3 1 0

↑

9 6 0

E t h o x y r e s o r u f i n

O - d e e

t h y l a s e

↓

2 3

↑

2 0

N D

↑

3 1 0

↑

3 1 4

P e n t o x y r e s o r t u f i n

O - d e p

e n t y l a s e

↑

1 0 8

↑ ↑

2 0

↑ ↑

2 0

↑

3 1 0

↑

3 1 4

T e s t o s

t e r o n e h y d r o x y l a t i o n

( t o t a l )

1 α -

h y d r o x y l a s e

2 α -


6 α -


7 α -


1 5 α

- h y d r o x y l a s e

1 6 α


2 β -


6 β -


1 5 β


1 6 β


A n d

r o s t e n e d i o n e

n o n e

n o n e

n o n e

n o n e

n o n e

↑

n o n e

↑ ↑

n o n e

↑ ↑

5 1 8 * *

1 0 8

*

↑ N D N D

n o n e

↑ N D

↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑

2 0 2 5 0

5 0 2 0 2 0 5 0 9 5 0 2 0

N D N D N D N D N D N D N D N D N D N D N D N D

n o n e

N D N D N D

n o n e

↑ N D

n o n e

n o n e

n o n e

n o n e

↑

* 3 5 0

n o n e N D N D N D

↑ ↑ N D

n o n e

n o n e

n o n e

n o n e

↑

* * *



S C C P / 1 1 9 2 / 0 8

O p

i n i o n o n

t r i c l o s a n

1 1 4

T a b l e

4 7 :

S u m m a r y o f E f f e c t s o n S e l e c

t e d B i o c h e m i c a l P a r a m e t e r s F o l l o w i n g 1 4 - D a y D i e t a r y A d m i n i s t r a t i o n o f T r i c l o s a n

M a

l e R a

t 1

M a

l e M o u s e

2

F e m a

l e M o u s e

2

M a

l e H a m s

t e r

3

F e m

a l e H a m s

t e r

3

E n

d - P o

i n t

E f f e c

t

M i n i m u m

E f f e c

t L e v e

l

( m g

/ k g

b w

/ d a y

)

E f f e c

t

M i n i m

u m

E f f e c t

L e v e

l

( m g /

k g

b w

/ d

a y

)

E f f e c

t

M i n i m u m

E f f e c

t L e v e

l

( m g

/ k g

b w

/ d a y

)

E f f e c

t

M i n i m u m

E f f e c

t L e v e

l

( m g

/ k g

b w

/ d a y

)

E f f e c t

M i n i m u m

E f f e c

t L e v e

l

( m g

/ k g

b w

/ d a y

)

C y t . P

/ - 4 5 0 p r o t e i n l e v e l s

C Y P

1 A

C Y P

2 B

C Y P

3 A

C Y P

4 A

↑ ↑ ↑

↑ ( s l . )

1 0 8 2 3 5 1 8

5 1 8

↓ N D

↑ ↑ ↑ ↑

5 0 2 0 2 0

↓ N D

↑ ↑ ↑ ↑

2 7 0

2 0 2 0

n o n e

N D

n o n e

↑

8 0 0

n o n e N D

↓

n o n e

3 1 4

1 R a t d a

t a f r o m M o l i t o r a n d P e r s o h n , 1 9 9 3 ( 1 6 9 ) .

2 M o u s e

d a t a f r o m M o l i t o r e t a l . , 1 9 9 2 ( 1 6 8 )

3 H a m s t e r d a t a f r o m T

h o m a s , 1 9 9 4 ( 1 7 0 ) .

* N o t s t a t i s t i c a l l y s i g n i f i c a n t b u t d o s e - r e s p o n s e e f f e c t o b s e r v e d .

N D , n o t

d o n e

s l . , s l i g h t



SCCP/1192/08


3.3.12.3.3 Summary of Liver Morphology and Biochemistry Data for Triclosan

The data showing strong increases in peroxisomal fatty acid beta-oxidation, 11- and 12-hydroxylation of lauric acid, and levels of CYP4A proteins (diagnostic for peroxisomeproliferation), and increases in numbers and size of peroxisomes provide biochemical and

EM evidence that triclosan has peroxisome proliferator-type activity in mouse liver at doses≥50 mg/kg body weight in males. In contrast, key events associated with PPARα agonists,such as lauric acid 12-hydroxylation and an increase in total cytochrome P450, weresignificantly increased in the high dose group in rats; however, the magnitude of theincrease was less than that seen in mice. The hamster data provide only limited evidence ofany peroxisome proliferator activities for triclosan, with increased CYP4A proteins and lauricacid hydroxylation activity occurring only at high doses [Thomas, 1994 (170)]. However,the lack of induction of peroxisomal fatty acid oxidation and of morphological evidencesuggests that triclosan is not a peroxisome proliferator in hamster liver.Mice and rats, but not hamsters, produced a dose-related increase in PROD, which wassignificant at all doses in the mouse [Molitor et al., 1992 (168)], but only significant in thehighest dose group in rats [Molitor and Persohn, 1993 (169)]. Activation of PROD is

typically associated with induction of CYP2B which is induced by phenobarbital. However, asis discussed more fully in Section 3.13.3, several other known PPARα agonists alsosignificantly increase induction of PROD. In examining the evidence for peroxisomeproliferator-like effects of triclosan in mice, it is interesting to note that the effects of theknown peroxisome proliferator clofibrate in rats treated for 3 days at the dose of 400 mg/kgbody weight/day were shown to include induction of PROD (3.7X), 4-nitrophenolhydroxylase (2.9X), and lauric acid hydroxylase (12.5X) activities compared to untreatedrats [Hanioka et al ., 1996 (171)].Overall, both biochemical and morphological evidence from special investigations of theeffects of triclosan in rodent liver serve to differentiate the response to triclosan in mice asthat of a peroxisome proliferator.

3.3.12.4. Effects of Triclosan on Rat Thyroid Effects of triclosan on thyroid hormone levels in rats have been investigated in two recentstudies, one with 4-day oral exposure (10, 30, 100, 300, 1000 mg/kg bw/d) in weanlingfemale Long-Evans rats (Crofton et al., 2007, AR4), the other with oral administration (3,30, 100, 200, 300 mg/kg bw/d) from postnatal day (PND) 23 to 53 in male Wistar rats(Zorilla et al., 2009, AR10).

Short term oral exposure in female rats resulted in dose dependent decreases in serumthyroxine levels: serum T4 was decreased 28, 34 and 53% following treatment with 100,300 and 1000 mg/kg bw/day triclosan, respectively. No significant changes were seen at10 and 30 mg/kg bw/day triclosan in female weanling rats. The authors of this study(Crofton et al. 2007) suggest that decreases in T4 may result from increases in the sulfationor glucuronidation via PXR-linked genes. This view is consistent with triclosan-induced up-regulation of liver enzymes documented in other studies that have been described above(section 3.3.12.3. and Tab. 3.3.12.3.2-1 and Tab. 3.3.12.3.2-2).

The purpose of the second study was to determine effects of triclosan on pubertaldevelopment and thyroid hormone levels in the male rat. After 31 days of exposure,triclosan significantly decreased serum thyroxine (T4) in a dose-dependent manner at 30mg/kg bw/day and higher (Zorilla et al., 2009). The active thyroid hormonetriiodothyronine (T3) was decreased significantly only at 200 mg/kg bw/day, and thyroidstimulating hormone (TSH) was not statistically different from controls at any dose. Liverweights were increased at 100 mg/kg bw/day triclosan and above suggesting that inductionof hepatic enzymes have contributed to the altered T4 and T3 levels. The authors did notconsider the levels of change in glucuronidation (UDPGT) activity at 30 mg/kg as sufficient

to explain the observed decrease in T4 levels; however, sulfation activity was not assessed.Triclosan did not alter the age at onset of puberty (assessed by preputial separation) or the



SCCP/1192/08


development of androgen-dependent tissues, even though there was a 60% decrease inandrogen serum levels in the 200 mg/kg dose group.

In conclusion, alterations in thyroid hormone levels induced by triclosan in juvenile malerats did not lead to any apparent functional consequences. Thus, the lowest observed effectlevel for a decrease in T4 (30 mg/kg bw/day) is regarded as biochemical effect marker, but

neither this nor the no observed effect level (3 mg/kg bw/day) are used for a riskassessment for triclosan since they have not been linked to an adverse effect.

Moreover, it is important to acknowledge major differences in the thyroid hormonephysiology and regulation between rats and humans (SCCNPF 2004, AR7). Since the rat isa very sensitive model for chemical induced changes in the thyroid hormone axis,limitations exist with respect to extrapolation of rat data to humanphysiology/pathophysiology.

3.3.13. Safety evaluation (including calculation of the MoS)

3.3.13.1 Consumer Exposure Assessment

For cosmetics, consumer exposure, measured as systemic exposure dose (SED) is typicallybased on dermal absorption data. In the case of triclosan, because of exposure throughtoothpaste use and mouthwash, oral exposure data are also relevant.

Calculations were made for individual products and for product groups in which triclosan isused according to the industry submission, i.e. most prevalently in toothpaste, deodorant,hand and body soaps (referred to as common-use products) and for products in whichtriclosan is used less frequently, such as facial cosmetic products, body lotion, andmouthwash (referred to as marginal-use products).For the purpose of SED calculations for oral formu lation s (toothpaste, mouthwash) it wasassumed that triclosan is 100% bioavailable (see Table 48).

In the dossier that was submitted, calculations were based on current-use triclosanconcentrations in different product types as given by the applicant, which, for some productcategories, are below the maximally allowed content of 0.3% triclosan. However, the SCCPwas requested to evaluate the safety of triclosan at the currently authorised level.Therefore, SEDs from both the current-use and the maximally allowed concentrations oftriclosan are given in the calculations below.

Table 48: SED Calculation for Oral Products

Product Assumedbioavailability

(%)1

Amountapplied

(mg)2

Retention3 Frequencyof

application(times/d)4

Triclosancontent

(%)

BW(kg)

SED(mg/kg

bw/d)5

Toothpaste 100 2,750 mgper day

0.17 NA 0.3 60 0.0234

Mouthwash 100 10,000 mgper use

0.1 3 0.26 60 0.1000

Mouthwash 100 10,000 mgper use

0.1 3 0.37 60 0.1500

Abbreviations: BW, body weight; d, day; NA, not applicable; SED, systemic exposure dose1 For the purposes of these calculations (i.e., for oral products), it was assumed that the bioavailability of

triclosan was 100%.2 Amount of application value was taken from Table 2, Section 6-2 in SCCP, 2006.3 Retention value was taken from Table 2, Section 6-2 in SCCP, 2006.4 Frequency of use per day value for toothpaste was taken from Table 3, Section 6-2 in SCCP, 2006, which takes

into account frequency of application. Frequency of use per day value for mouthwash was taken from Table 2,Section 6-2 of the SCCP, 2006 guidance.



SCCP/1192/08


5 Formula: SED = (Bioavailability in % x amount of product applied (mg) x Frequency x Retention factor xamount of triclosan in product) / BW.

6 Current use concentration as given by the applicant7 maximally authorised concentration

For dermal formulations, SED calculations were based on percutaneous absorption datafrom in vitro human studies (Table 37). SED calculations for individual personal-careproducts containing triclosan were carried out based on dermal absorption values (µg/cm2)from in vitro percutaneous absorption studies conducted with deodorant and w/oformulations containing 0.2% triclosan and dilute soap solution formulation containing0.02% triclosan.In each calculation for dermal products, an extrapolated value for flux (µg/cm2 absorption)was used, based on the assumption that skin penetration is by passive diffusion, such thatflux would be proportional to the concentration of triclosan applied to the skin. For handsoap and body soap, the conversion of the µg/cm2 dermal absorption value to a current-usevalue for amount of triclosan in the product type assumed a 10-fold dilution of 0.3%triclosan. For both soaps, a retention factor was not used due to the inclusion of a rinse-offstep in the relevant in vitro percutaneous absorption study. The results of thesecalculations are provided in Tables 49 and 50, below, for leave-on and rinse-off products,

respectively.

Table 49: SED Calculation for Leave-On Products

Product 24-hdermal

absorption

based on

0.2%triclosan

(µg/cm2) 1

Triclosancontent

(%)

Calculated24-h dermal

absorption

based on

triclosancontent

(µg/cm2) 2

SA(cm2)

3

F(per

d)

Conver-sion

(mg/µg)

R BW(kg)

SED(mg/kg

bw/d)5

Deodorantstick

0.303 0.3 0.455 200 1 1x10-3 1 60 0.0015

BodyLotion

0.420 0.156 0.315 15670 1 1x10-3 1 60 0.0823

BodyLotion

0.420 0.37 0.630 15670 1 1x10-3 1 60 0.1646

Facepowder

0.420 0.26 0.420 565 1 1x10-3 1 60 0.0040

Facepowder

0.420 0.37 0.630 565 1 1x10-3 1 60 0.0060

BlemishConcealer

0.420 0.156 0.315 57 1 1x10-3 1 60 0.0003

BlemishConcealer

0.420 0.37 0.630 57 1 1x10-3 1 60 0.0006

Abbreviations: BW, body weight; d, day; F, frequency of application; h, hour; R, retention; SA, surface area ofapplication; SED, systemic exposure dose1 Dermal absorption values based on in vitro data using 0.2% in deodorant formulation (for deodorant stick) and

0.2% water/oil emulsion (for body lotion, face powder, and stick concealer).2 Calculation: (Absorption from 0.2% triclosan applied in the relevant in vitro study) x (triclosan content for the

product/0.2%) = Absorption from 0.3% triclosan. This assumes that skin penetration is by passive diffusion,such that flux would be proportional to the concentration of triclosan applied to the skin.

3 Area of application values were taken from Table 1, Section 6-2 in SCCP, 2006. The skin area for blemishconcealer was assumed to be 1/10th of the face.

4 Frequency of application values for use per day were taken from Table 2, Section 6-2 in SCCP, 2006.Frequency of application values for face powder and stick concealer were assumed to be once per day.

5 Formula: SED = (24-h dermal absorption in µg/cm2 based on use levels of triclosan in product x surface area ofapplication in cm2 x frequency of application per day x retention x conversion factor) / BW.

6 Current use concentration as given by the applicant7

Maximally authorised concentration



SCCP/1192/08


Table 50: SED Calculation for Rinse-Off Products

Product 24-hdermal

absorption

based on0.02%

triclosan

(µg/cm2) 1

Calculated24-h

dermal

absorptionbased on0.03%

triclosan

(µg/cm2) 2

SA(cm2) 3

F(times/d) 4

Conversion(mg/µg)

BW(kg)

SED(mg/kg

bw/d)5

Hand soap 0.0306 0.046 860 10 1x10-3 60 0.0066

Showergel/bodysoap

0.0306 0.046 17,500 2 1x10-3 60 0.0268

Abbreviations: BW, body weight; d, day; F, frequency of application; h, hour; SA, surface area of application; SED,systemic exposure dose1 Dermal absorption value based on in vitro data using 0.02% soap solution.2 Calculation: (Absorption from 0.02% triclosan applied in the relevant in vitro study) x (0.03%/0.02%) =

Absorption from 0.03% triclosan solution. This assumes that 1) a 10X dilute solution of 0.3% triclosan is

applied and 2) skin penetration is by passive diffusion, such that flux would be proportional to the concentrationof triclosan applied to the skin.

3 Area of application values were taken from Table 1, Section 6-2 in SCCP, 2006.4 Frequency value for shower gel/body soap use per day was taken from Table 2, Section 6-2 in SCCP, 2006.

Frequency for hand washing was not provided in SCCP, 2006, so was assumed to be 10 times per day for thiscalculation.

5 Formula: SED = (24-h dermal absorption in µg/cm2 based on 0.03% triclosan x surface area of application incm2 x frequency of application per day x conversion factor) / BW.

The combined SED for common-use triclosan-containing personal care products andmarginal-use triclosan-containing personal care products was also calculated. The SEDcalculations are presented in the Table below.

Summary of Triclosan SED Values from the Use of Personal Care Products

Type of Product(s) Triclosancontent (%)

SED

Toothpaste 0.3 0.0234

Hand Soap 0.3 0.0066

Body Soap/shower gel 0.3 0.0268

Deodorant (Stick) 0.3 0.0015



Face powder 0.2 0.0040Face powder 0.3 0.0060



Stick-type concealer 0.15 0.0003

Stick-type concealer 0.3 0.0006

Common-Use Products (toothpaste, handsoap, body soap/shower gel, deodorant stick)

0.3 0.0583

Marginal-Use Products (mouthwash, bodylotion, face powder, stick concealer)

0.15-0.2 0.1866

Marginal-Use Products (mouthwash, body

lotion, face powder, stick concealer)

0.3 0.3212

All Products (toothpaste, hand soap, body 0.15-0.3 0.2449



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Summary of Triclosan SED Values from the Use of Personal Care Products

Type of Product(s) Triclosancontent (%)

SED

soap/shower gel, deodorant stick,mouthwash, body lotion, face powder, stickconcealer)

All Products (toothpaste, hand soap, bodysoap/shower gel, deodorant stick,mouthwash, body lotion, face powder, stickconcealer)

0.3 0.3795

Abbreviations: SED, systemic exposure dose

Internal exposure and absorption of triclosan under simulated use conditions by humans canbe approximated also from in vivo studies with volunteers that applied triclosan-containingpersonal care products for a prolonged time period (see table 36). Precise information onthe use-pattern and the level of triclosan content of the formulations was available in thestudy by Beiswanger and Tuohy (1990), in which 182 subjects used a toothpaste (0.28 %triclosan), a bar soap (0.75% triclosan) and a deodorant (0.39% triclosan) for 13 weeks.

The results of this study indicated that all subjects reached a stable plateau plasma levelafter 3 weeks of use of the toothpaste, deodorant, and soap. The results showed plasmalevels of: 19-23 ppb (exposure to toothpaste only), and 29-31 ppb (exposure to toothpaste,deodorant, and soap).

3.3.13.2 Safety Assessment

The human plasma levels can be compared to the plasma level (of 28,160 ng/ml) reportedin studies with rats that received triclosan doses at the NOAEL of 12 mg/kg bw/day (seeTab. 27) to derive plasma level based MoS.

Types of Products Used SED

(mg/kgbw/d)

MoS

Based on RatNOAEL of

12 mg/kg bw/d

MoS

Based on PlasmaLevels

Toothpaste 0.0234 513 1408

Toothpaste, deodorantstick, and hand soap

0.0315 381 939

Common-Use Products0.3% triclosan (toothpaste,hand soap, bodysoap/shower gel,deodorant stick)

0.0583 206 Not done(no human plasma

dataavailable)

All Products0.15 – 0.3% triclosan(toothpaste, hand soap,body soap/shower gel,deodorant stick,mouthwash, body lotion,face powder, blemishconcealer)


dataavailable)

All Products0.3% triclosan(toothpaste, hand soap,body soap/shower gel,deodorant stick,mouthwash, body lotion,

face powder, blemishconcealer)


dataavailable)



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From the calculations above it can be deduced that the use of triclosan in all products is notsafe because of the magnitude of the aggregate exposure. This conclusion is reachedregardless whether current use levels as given by the applicant or the maximally authoriseduse level is considered. However, the use in the common-use products (toothpaste, handsoap, body soap/shower gel and deodorant stick) is considered safe. The exposure to

triclosan from face powder and blemish concealer (up to 0.3% triclosan) is low and notconsidered to be of concern in addition to exposure from common-use products. However,the use of triclosan in body lotions and mouthwashes results in high exposures and is notrecommended.

Safety of Triclosan in Children and Neonates

The very low levels of exposure as measured in breast milk indicate that maternal use oftriclosan immediately post-partum is unlikely to be a safety concern for neonates (see3.3.11.2.6). Infant exposure to triclosan from breast milk has been shown to besignificantly lower than exposure to the mother, based on a comparison of triclosanconcentrations in breast milk and plasma [Allmyr et al ., 2006 (103)].

No measured exposure data for babies and young children following use of consumerproducts containing triclosan was identified in the literature, except spot urinemeasurements in the age group 6-11 years from the NHANES study [Calafat et al. 2008;AR3]. Based on the conversion of spot urine concentrations to estimated dose, thissubpopulation had a lower aggregate exposure to triclosan than children of 11-19 years andadults [US EPA 2008 (AR9)]. The rapid increase in maturation of glucuronidation abilitywithin the first year and the maturity of the sulfation pathway, indicate that capabilities ofchildren to metabolise triclosan through glucuronidation or sulfation are likely comparable tothose of adults. Also, glomerular filtration rates normalised to body weight approach adultvalues by around 6 months of age and renal tubular function matures to near-adult valuesby around 1 year of age (Alcorn and McNamara, 2002). Accordingly, studies have shownthat elimination is comparable in adults and children (see Section 3.11.2.1).

3.3.14. Discussion

Physico-chemical properties

Trichlosan is a phenol and a weak acid (pKa 8.1). This and its partition coefficient (logPo/w4.8) facilitate transfer of the protonated (non-ionized) form of triclosan across lipidmembranes.

General toxicityTriclosan is not acutely toxic via the oral route of administration, with high oral intubationLD50 values in the range of 3,750 to 5,000 mg/kg body weight in mice and rats, and anoral capsule LD50 value of greater than 5,000 mg/kg body weight in dogs. SCCP considers

the NOAEL as 12 mg/kg bw/d due to haematoxicity and decreased absolute and relativespleen weights (Mid Dose Females) in the long term toxicity study in rats.

Irritation / sensitisation

The irritation/corrosivity data from either irritation studies in the hamster, guinea pig, andrabbit, or skin toxicity studies conducted in the mouse, rat, monkey, and dog suggest thattriclosan may cause slight reversible skin irritation at concentrations of 0.5 to 5% underexperimental conditions. Triclosan at concentrations of 1 to 10% produced only slight,reversible irritation in the rabbit eye. Data from human use evaluating the skin and oralmucosa irritation effects of triclosan alone, or in combination with SLS, indicate thattriclosan 0.3% is not a skin or oral mucosal irritant.In the guinea pig no sensitisation with triclosan in various formulations and concentrations(up to 10% in petrolatum) was found. However, clinical experience has shown that triclosandoes have a low sensitisation potential in humans. In over 14,000 patients patch testedwith triclosan (typically tested at a concentration of 2% in petrolatum), the range of positive



SCCP/1192/08


results was 0.1 to 0.3% of the tested groups. When tested in patients with known orsuspected cosmetic allergy or intolerance positive reaction rates ranged from 0.06 to 0.8%of a total of 11,887 tests conducted. Possible photocontact allergy has been rarely reported.

Dermal and oral absorption

Data from percutaneous absorption studies indicate that triclosan is well absorbed through

the skin in all species tested with the extent of absorption being dependent on theformulation in which it was delivered. In the rat, percutaneous absorption wasapproximately 23 to 28% of the applied dose of triclosan in ethanol, ethanol/ water, soapsuspension, or a cream formulation.Triclosan is highly absorbed following oral administration, with no species-relateddifferences, and in humans this is up to 98% of the dose. However, under normal conditionsof toothpaste use (i.e., expectoration and rinsing) or following percutaneous application ofseveral different personal care products, absorption is more limited (approximately 5 to10% of the dose via either of these routes of administration).See also brief summary of in vivo data under “Kinetics”

Mutagenicity / genotoxicity

The genotoxic potency of triclosan has been investigated in a number of tests which can bebroadly sub-divided in non-regular and normal (regulatory accepted) tests. Most of thetests are rather old and performed before the introduction of OECD guidelines.Consequently, the latter tests are not performed under currently accepted protocols. Since,next to non-standardised protocols, the tests have limited value but may occasionally givesupportive evidence. Only two of the (non-regular) tests indicate a putative genotoxicpotential of triclosan: Irgasan DP 300 (triclosan) induced mutations in an in vitro genemutation assay in yeast. This positive result is not confirmed in an appropriate genemutation test in mammalian cells. The same compound also induced mutations in a mousespot test. However,, in a similar experiment with lower and thus less toxic concentrationsthis result could not be confirmed.

Triclosan was investigated in (regular) genotoxicity tests covering the 3 endpoints: gene

mutations, structural and numerical chromosome aberration. Triclosan exposure did notresult in gene mutations in bacteria or mammalian cells nor did it induce UDS in vitro inprimary hepatocytes. Triclosan induced chromosome aberrations in V79 cells, but wastested negative in assays with CHO cells. The positive result could not be confirmed in an invivo micronucleus test in bone marrow cells of mice. Consequently, triclosan can beconsidered to have no relevant genotoxic potential in vivo.

Carcinogenicity

Three rodent lifetime bioassays have been conducted to evaluate the carcinogenic potentialof triclosan. Triclosan produced hepatic effects and hepatic tumours in mice, but littleevidence of toxicity and no tumours in rats. Hamsters showed increased liver toxicityrelative to the rat, but no tumours.

According to the EU classification system, triclosan is not considered classifiable as acarcinogen. It should be noted that triclosan is a peroxisome proliferator in mice liver.

Reproductive/developmental ToxicityTriclosan was not teratogenic nor a reproductive toxicant in a full complement ofreproductive and developmental toxicity studies conducted in mice, rats, and rabbitsconducted at doses of up to 350 mg/kg body weight/day.

NOAEL (NOEL) values from the definitive GLP studies were summarized in Table 21. It isimportant to note the determination of the foetal NOAEL value for each study was based onfoetal variation effects that were most likely secondary to general maternal toxicity, and notdirect effects of triclosan per se. It is also worth noting that the low NOAEL value for foetal

effects in the mouse study (25 mg/kg body weight/day) is likely attributable to thesensitivity of the maternal mice to the liver effects of triclosan, also observed in therepeated dose and carcinogenicity studies in mice.



SCCP/1192/08


Kinetics

Numerous human and animal studies are available on the toxicokinetics of triclosanfollowing oral and dermal exposure to single and repeated doses. The studies cover allimportant aspects, i.e. absorption, distribution, metabolism and excretion. Upon oraladministration absorption of triclosan from the gastrointestinal tract is rapid and extensive

in both humans and animals. But, limited buccal absorption was seen in humans followingnormal toothpaste use (up to 14% of the amount that would be absorbed upon ingestion ofan equivalent dose). Upon dermal application in humans, absorption was at least 3% to 7%,and at least 14% in one volunteer.Triclosan is rapidly distributed in the organism following oral or dermal exposure. The mainmetabolic pathways in humans and animals involve glucuronidation and sulfation by phase-2 enzymes. The half-life of elimination for orally administered triclosan ranged from 13 to29 h in humans compared to 10 to 15 h in rats, 8-12 in mice and 25 to 32 h in hamsters.The major route of excretion in humans, hamsters, rabbits and primates is via urine, withexcretion via faeces being of secondary importance in these species. The reverse situation isobserved in rats, mice and dogs where biliary excretion is more important than renalexcretion. The human oral and dermal data provide no evidence for a bioaccumulation

potential. Likewise, the kinetic data in rats and hamsters provide no evidence for abioaccumulation in these species, whilst in mice retention of triclosan (and/or metabolites)appears to occur in liver.In conclusion, kinetics of triclosan are qualitatively similar, but the observed quantitativedifferences between humans and several animals make human data the first choice for thesafety evaluation of triclosan-containing consumer products.

Other aspectsRecently, the US EPA (2008) utilized population-based biological monitoring data fortriclosan (available from the NHANES study) to assess the co-occurrence of uses to developan aggregate exposure assessment. Because of some uncertainties in converting spot urine

concentrations to estimated dose, three conversion methods were used. Calculatedexposure was then compared to the selected oral NOAEL of 30 mg/kg/day (from the chronictoxicity study in baboons). Based on the results at the mean and 99 th percentile, theaggregate risks to triclosan from all (personal care and other consumer products) uses didnot trigger a risk of concern. The mean MOEs ranged from 4,700 to 19,000. The MOEs atthe 99th percentile ranged from 260 to 1,500 [US EPA 2008, AR9].

Exposure estimates based on biological monitoring data from the US are considered bySCCP as useful additional information in their overall evaluation on the safety of triclosan.

The difference in SCCP and US-EPA evaluations of triclosan may be explained as follows:

- USA-EPA chose a NOAEL of 30 mg/kg/d whereas SCCP selected a NOAEL of 12 mg/kg/d(based on haemotoxicity) as the critical effect level against which human exposure totriclosan is compared (for subsequent MOE or MOS calculations). The SCCP approach is inline with the evaluation of triclosan by EFSA for its use in food contact materials.- US-EPA has estimated triclosan exposure in the US population on the basis ofbiomonitoring data from spot urine samples. Although this approach probably reflectsexposure from current use concentrations in various products on the US market, it cannotbe applied directly to the evaluation regarding the safe use of triclosan in cosmetic productsby SCCP, since:

- The current use concentrations in the USA may have been lower than the maximaltriclosan concentration limit of 0.3% as preservative in cosmetic products in the EU,the safety of which SCCP was asked to evaluate according to this mandate (Question1)

- In estimating human exposure, the SCCP followed its Notes of Guidance to calculatesystemic exposure doses (SED) from triclosan-containing products (at 0.3%) applied



SCCP/1192/08


orally and dermally. This may be viewed as a worst-case scenario. The alternativeapproach, i.e. MOS calculations that are based on plasma levels (measured undersimulated use-conditions) were only available for certain products, not for all triclosan-containing products. Representative biomonitoring data are not available for theEuropean population.

It is important to note that the two evaluations followed different objectives: While US-EPAin principle looked at real exposure that occurred in the population to derive a conclusionabout a possible concern, the SCCP is asked to evaluate the safety of a hypotheticalmaximum exposure according to the authorised concentrations and applications in thecosmetic legislation.

4. CONCLUSION

Taking into account the provided toxicological data, the SCCP considers that the continueduse of triclosan as a preservative at the current concentration limit of maximum 0.3% in all

cosmetic products is not safe for the consumer because of the magnitude of the aggregateexposure.

However, its use at a maximum concentration of 0.3% in toothpastes, hand soaps, bodysoaps/shower gels and deodorant sticks ("common-use products" as defined by theapplicant) is considered safe. Any additional use of triclosan in face powders and blemishconcealers at this concentration is also considered safe but the use of Triclosan in otherleave-on products (e.g. body lotions) and in mouthwashes is not considered safe for theconsumer due to the resulting high exposures.

Importantly, before a final conclusion on the safety of triclosan in cosmetic products can bereached, the potential development of resistance to triclosan and cross-resistance by certain

micro-organisms must be assessed. This aspect is not covered in this document and will bediscussed in a separate opinion.

Inhalation exposure to triclosan from spray products (e.g. deodorants) was not assessed.

5. MINORITY OPINION

Not applicable

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Additional references added by SCCP

AR1. Allmyr M, Harden F, Toms LM, Mueller JF, McLachlan MS, Adolfsson-Erici M,Sandborgh-Englund G (2008). The influence of age and gender on triclosanconcentrations in Australian human blood serum. Sci Total Environ. 393(1):162-7.

AR2. Calafat AM, Ye X, Wong LY, Reidy JA, Needham LL (2008). Urinary concentrations oftriclosan in the U.S. population: 2003-2004. Environ Health Perspect. 116(3):303-7.

AR3. Campbell L, Zirwas MJ. (2006). Triclosan. Dermatitis 17(4):204-7.

AR4. Crofton KM, Paul KB, DeVito MJ, Hedge JM (2007). Short-term in vivo exposure tothe water contaminant triclosan: evidence for disruption of thyroxine. EnvironmentalToxicology and Pharmacology 24: 194-197.

AR5. Danish Environmantal Protection Agency (2007). Survey and risk assessment ofchemical substances in deodorants. Survey of Chemical Substances in ConsumerProducts, No. 86 http://www2.mst.dk/Udgiv/publications/2007/978-87-7052-625-8/pdf/978-87-7052-626-5.pdf

AR6. EFSA (2004). Opinion of the Scientific Panel on food additives, flavourings,processing aids and materials in contact with food. The EFSA Journal 37, 1-7

AR7. SCCNFP (2004). Opinion concerning Iodopropynyl carbamate (P91). Adopted by theSCCNFP on 1.7.2004http://ec.europa.eu/health/ph_risk/committees/sccp/documents/out288_en.pdf

AR8. SCF (2000). Opinion of the Scientific Committee on Food on the 10th additional listof monomers and additives for food contact materials (adopted by the SCF on22/6/2000) http://europa.eu.int/comm/food/fs/sc/scf/out62_en.pdf

AR9. United States Environmental Protection Agency (2008). 5-Chloro-2-(2,4-dichlorophenoxy)phenol (Triclosan): Risk Assessment for the Reregistration EligibilityDecision (RED) Document. Case No 2340. (Docket EPA-HQ-OPP-2007-0513; PCCode: 054901. DP Barcode: 373535) available under http://www.regulations.gov

AR10. Zorilla LM, Gibson EK, Jeffay SC, Crofton KM, Setzer WR, Cooper RL, Stoker TE(2009). The effects of triclosan on puberty and thyroid hormones in male Wistar rats.Toxicological Sciences 107: 56-64.



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EUROPEAN COMMISSIONHEALTH AND CONSUMERS DIRECTORATE-GENERAL

Public Health and Risk Assessment

Risk assessment

Brussels,SANCO.C7

EXPLANATORY NOTE FOR THE MODIFICATIONS OF THE SCCS OPINION ON

TRICLOSAN (ANTIMICROBIAL RESISTANCE) FOLLOWING THE PUBLIC

CONSULTATION ON THE DRAFT FINAL OPINION

This note sets out the rationale for the modifications made to the opinion of the European

Commission Scientific Committee on Consumer Safety (SCCS) on triclosan (antimicrobialresistance) following a public consultation conducted between 29 March 2010 and 26 May 2010.

Introduction

In May 2009, the European Commission requested the Scientific Committee on Consumer Safety

to assess the effects of triclosan on the emergence of bacterial resistance. A SCCS WorkingGroup comprising of a member of the SCCS, a member of the Scientific Committee on Emerging

and Newly Identified Risks (SCENIHR), a member of the Scientific Committee on Health and

Environmental Risks (SCHER) and an expert from academia with experience on the subject wasformed. The WG produced a draft opinion which was discussed and adopted by the SCCS

plenary on 23 March 2010 as a preliminary opinion suitable for public consultation.

In line with its procedures for stakeholder dialogue, implemented in the Rules of Procedures of

the new Scientific Committees set up by Commission Decision 2008/721/EC of 5 September2008, the European Commission Health and Consumers Directorate General (DG SANCO)

conducted a public consultation on the preliminary opinion of SCCS between 29 March and 26

May 2010.

Results/participation

By the deadline, DG SANCO received a total of 10 contributions. All of them were reviewed by

the Working Group during its meeting on 8 June 2010 and appropriate modifications wereintroduced into the opinion which was then discussed and adopted as the final opinion by the

SCCS at its plenary of 21 June 2010.

Modifications to the opinion

The opinion has been modified to take into account all submitted comments which were assessed

by the Working Group to be pertinent and relevant for the subject matter and which were within




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the competences of the Scientific Committees and respected the clear separation between riskassessment and risk management that underpins the Scientific Advisory structure of the European

Commission. Comments on policy, risk management, legal clarification, ethics, the precautionary

principle, were not considered as, although pertinent to the subject matter, they are outside thecompetences of the Scientific Committees.

Detailed explanations of the way the comments received were treated by the SCCS are provided below. The numbering of paragraphs and lines correspond to the sections of the final opinion

adopted by the SCCS on the 21 June 2010 and published together with this document.

Changes to the opinion

1. On page 16, section 5.2, a sentence reporting on the reference Bojar et al. (2009) was added

as a complement of information.

2. On page 26, section 6.3, a sentence reporting on the reference Bayston et al. (2007) was

added. This reference reinforces the evidence base presented in the section on by-pass metabolic

blockage.3. On page 36, with respect to the negative results from the in situ studies, the text was

amended as follows: "While these results are at first sight reassuring, the differences of

methodologies used to measure “resistance” and to analyse the data make it premature at thisstage to conclude that triclosan exposure never leads to developing microbial resistance. In

addition, these useful in situ studies do not provide information on expression of genes involvedin resistance, maintenance of resistance and virulence genes and transfer of resistance

determinants. Thus this opinion strongly recommends to perform additional in situ studies

looking at these aspects and bacterial phenotypes where known concentrations of triclosan have been found in the environment."

4. On page 30, section 6.6.2, text on the reference Pycke et al was added: "Pycke et al. (2010)

observed that triclosan exposure of the environmental α-proteobacterium Rhodospirillum rubrum

led to an increase in triclosan MIC. The extent of this increase as well as the generation ofdifferent antibiotic susceptibility profiles was triclosan-concentration dependent, indicating the

expression of distinct resistance mechanisms." Triclosan resistance is rarely found in clinical

strains because it is rarely looked for.

5. On page 27, section 6.3, the following text was added: "It is however interesting to note thatTabak et al. (2009) observed a synergistic action of sequential treatment of triclosan (500 µg/ml)

followed by ciprofloxacin (500 µg/ml) against biofilm of S. enterica serovar Typhimurium. There

is little information in the literature about the potentiation of activity between a biocide and an

antibiotic and such a study is important and provides interesting application/effect of triclosan."

6. The reference of Cottell et al is already mentioned in the text, page 30.

7. On pages 37-38, the text of the opinion (section 12) has been modified to clarify the use of in

situ data in relation to in vitro investigations.

8. On page 29, section 6.6.2, text referring to Pycke et al (2010) on the characterization of

triclosan-resistant mutants was added

9. On page 26, section 6.3, text referring to Yu et al (2010) on the signature gene expression




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profile of triclosan-resistant Escherichia coli was added.

10. On page 25, section 6.3, text referring to Zhu et al (2010) on triclosan resistance of Pseudomonas aeruginosa was added.

11. On page 27, section 6.3, text referring to Tabak et al (2009) on the synergistic activity of

triclosan and ciprofloxacin on biofilms of Salmonella Typhimurium was added.

12. The conclusion of the opinion has been clarified to reflect comments received suggestingadditional clarity.

Comments for which no changes could be made

In addition to the comments received which resulted in the above changes, the following

comments were received and were evaluated by the SCCS but no changes were introduced in theopinion. The main reasons for this are : 1) comments were outside the scope of the terms of

reference for this opinion; 2) comments were outside the competences of the Scientific

Committees (and SCCS in this case) as they concerned policy, risk management, on field use of

DU ammunition and on site sampling and surveillance; 3) in the scientific judgement of theSCCS, the submitted scientific evidence and argumentation were not of sufficient quality and

strength to support changes and modifications in the opinion and its conclusions. For reasons of

clarity, a brief rationale underpinning its evaluation of each comment is provided for eachcomment.

1. Regarding the comment on stability and solubility of triclosan, the normal storageconditions are not defined in the submission. Quantitative data on the solubility of triclosan in

DMSO are not available. The SCCS considers that triclosan is soluble in DMSO. This opinion is

not dealing with the ecotoxicity of triclosan.

2. Regarding the comment on evidence of the potential of triclosan to induce or transmit

antibacterial resistance stating that since 2006 "there do not appear to be any compelling

reasons or scientific data to support different conclusions regarding the potential for triclosan toinduce or transmit antibacterial resistance. In fact, there are several studies that provide support

for a lack of antibacterial resistance in situ (Cole et al. 2003; Jones, 2000; Ledder et al. 2006; McBain et al. 2004; Sullivan et al. 2002)", the WG agrees. This is actually in the text of the

opinion in section 5.5.2.

3. The WG agrees on the lack of standardized methods for MIC determination. These pointsare covered in the document. There is no recognized bacterial model for the study of biocide

resistance (SCENIHR, 2009).

4. Regarding the comment on the in situ clinical and environmental studies that "the 2010

SCCS draft document should give more consideration to the in situ clinical and environmental

studies of triclosan and its impact on antimicrobial resistance as recommended by Russell(2004)", the SCCS considers that the information provided by Russell (2004) is limited and

largely agrees with the data and conclusion of the opinion.

5. The SCCS considers that number of in situ studies conducted is limited. In addition, themethodologies used in these studies differ, notably in the measurement of resistance and although

useful, the data provided by these studies showing a lack of correlation between triclosan usage

and selection of triclosan resistance are limited. In particular, they do not incorporate any genetic




Triclosan p.4

aspect of resistance. Therefore, the SCCS concluded that it is still not possible to quantify thegenetic risk associated with triclosan usage (and any other biocide) and recommended in its

opinion that additional studies be performed.

6. The in situ studies focused on measuring triclosan resistance and antibiotic resistance

following triclosan exposure. They did not look into the expression of genes involved in

resistance, maintenance of resistance and virulence genes and transfer of resistance determinants(SCENIHR 2010). The six in situ studies reported in this opinion showed no increase in bacterial

resistance following exposure to triclosan. However, in these studies, not all hazards (e.g. geneticaspects) have been measured.

7. The points regarding dental plaque and gingival health and the lack of resistance found in a

number of environmental isolates are already covered by the opinion. The SCCS considers that:

• The status of gingival health is not an indication of the lack of bacterial resistance.

• While the Walker study and the other studies cited were state-of-the art at the time theywere performed, they did not have the modern tools (e.g. proteomic or genomic analysis)

available today to investigate the complete bacterial population and the bacterial responseto biocides.

8. Regarding the comment according to which triclosan-induced antibacterial resistance has

not been convincingly demonstrated in the group of in situ studies discussed, the SCCS is of

the following view. Only few in situ studies have been conducted. Through the use of differentmethodologies and analysis of data, these studies did not find a correlation between triclosan

exposure and emerging resistance. This contrasts with studies performed in vitro and emphasizes

the need for translational research. The development of bacterial resistance through well-definedmechanisms, notably following triclosan exposure, has been very well-described. In situ studies

have only focused on MIC and cross-resistance to antibiotics and demonstrated a lack of both

following triclosan exposure. These studies have however not looked at the phenotypic

expression of these mechanisms, nor at the maintenance of the gene pool and transfer ofresistance determinants. With this in mind, the information obtained in situ is limited, and it is

premature at this point to conclude that triclosan is not of any concern. It must be emphasized

that, although this opinion focuses on triclosan, the conclusion and observation drawn in thisdocument are also valid for other biocides.

9. Regarding the comment that the statement that environmental concentrations in selected

geographical regions are high enough that “triggering of bacterial resistance could also

occur in the environment” is speculative and not supported by the studies conducted by Ledderet al. (2006) and McBain et al. (2004), the SCCS is of the following view. The McBain et al.

(2004) study is an in vitro study. The Ledder et al. (2006) is an ex situ study and actually showed

that bacterial microcosm exposure to triclosan did not result in widespread high level resistance,

except for enteric bacteria, especially E. coli. The McBain et al. (2003) ex situ investigationactually highlighted a change in bacterial microcosm composition although the overall

microcosm resistance to triclosan as measured by an increase in MIC did not change. Again, thisis not unique to triclosan. Where a selective pressure exerted by a biocide is present, then

alteration of a microcosm is to be expected.

10. The SCCS considers that reports investigating triclosan low level resistance in clinical

Acinetobacter strains (Chen et al. 2009) should be taken seriously and further research should be




Triclosan p.5

conducted on the mechanisms and conditions leading to increase of resistance in the environmentis already mentioned in the text (reference to Chen et al, page 25).

11. The SCCS considers the comment that environmental conditions favourable for induction

of triclosan resistance is an interesting point and that it should be considered in future research

projects as previously mentioned in the SCENIHR (2010) opinion on biocides research.

12. The SCCS agrees with the comment on limiting the use of triclosan without proven benefit

for human health but also accepts however, that where evidence exists that triclosan use in

beneficial in e.g. preventing disease in humans, it should be encouraged. Hence prudent use is

mentioned in the conclusion of the opinion: When used appropriately, biocides, includingtriclosan, have an important role to play in disinfection, antisepsis and preservation.

13. Concerning the comment on the difference between the previous SCCP opinion and this

one by the SCCS , the main difference is the scientific information available on proteomic and

genetic aspects of triclosan resistance.

14. This opinion is based on the weight and quality of the available scientific evidence regardlessof its source. On that basis, the SCCS notes that the lack of resistance reported in some in situ

studies did not take into account the recent developments in genetic and proteomicmethodologies.

15. Concerning the comments on the value of the comparative study by Lambert (2004) withclinical isolates of methicillin-resistant and methicillin-sensitive Staphylococcus aureus and

Pseudomonas aeruginosa which showed no indications for a connection between triclosan

resistance and antibiotic resistance under real-world conditions, the views of the SCCS are the

following: this was a retrospective study looking at the MIC profile of various clinical isolates to

a range of biocides. There were indeed no differences in susceptibility of the clinical isolates tovarious biocides over time. This study confirmed that antibiotic resistant bacterial isolates might

not necessarily be less susceptible to a range of biocides. However, in the view of the SCCS, this

study did not inform on the continuous use of biocides on antibiotic resistance in the clinicalsettings. This view was first formulated by Russell in 2002.

16. Concerning comments on the continued use of triclosan, due to the limited number of in

situ studies of resistance induced by triclosan to date, the SCCS can only recommend the prudent

use of triclosan, for example in applications where a health benefit can be demonstrated.However, the SCCS considers that conclusions from in vitro studies cannot be ignored, notably,

the role of triclosan (and other biocides) in triggering resistance and in the dissemination (or lack

of) resistance determinants. Hence, the SCCS appreciates that research investment from industrywill be maintained to contribute to a better understanding of the potential risks associated with

triclosan applications. Research in triggering mechanisms of resistance, maintenance of the gene

pool and the transfer of resistance and virulence determinants, and improving the translational

application of laboratory results to situations in situ are needed. In that spirit, the SCCSappreciates the comments received that product manufactures are taking approaches which limits

the use of triclosan to a limited number of products with a demonstrated health benefit.

17.Comemnts on ecotoxicity were not considered as they are out of the scope of this opinion.

18. With respect to comments on the essential differences between the concepts of resistance

to antibiotics and resistance to disinfectants (Cerf et al 2010), the SCCS will point to the 2009

SCENIHR opinion on the antibiotic resistance effect of biocides.




Triclosan p.6

19. Concerning the comments on the significance of the Chen et al (2009), and Stickler and

Jones (2008) studies the views of the SCCS are the following: These are not in situ studies. Chen

et al. 2009 observed that the majority of the A. Baumannii was susceptible to triclosan with only

3% showing reduced susceptibility (max. 16 mg/L). They further observed that all triclosanresistant isolates were also resistant to important chemotherapeutic antibiotics while the

susceptible isolates showed a resistance percentage between 40% and 55%. Stickler and Jones(2008) has been cited several times in the opinion.

20. Concerning comments on the link between triclosan exposure and resistance to important

antibiotics in vitro this has been made clear in the opinion. The role of biocides, including

triclosan, in emerging resistance to clinically effective antibiotics and their impact in clinical

settings needs to be clarified. In the views of the SCCS, this is certainly of a lesser concern thanthe improper use of antibiotics in clinical settings triggering resistance as mentioned in the

SCENIHR 2009 opinion.

21. Concerning the comments on the use concentrations of triclosan, the information available

to the SCCS for the elaboration of its opinion are from 2007. The SCCS has no information onthe triclosan use patterns before 2007 and therefore is not in a position to establish a link between

the environmental concentrations of triclosan and its use in cosmetic products only.

22. Concerning the comment on soil bacteria, the opinion (section 10.2) makes clear that theexposure to some biocides favours the dissemination and maintenance of genetic mobile

elements. However, there is no such information available concerning triclosan. On that basis, the

SCCS in it opinion (section 10.3), formulates a number of questions that need to be answered to be able to perform a risk assessment on this issue.

23. The comment that the environmental concentrations of triclosan depend upon the

efficiency of WWTPs is already stated in the opinion (section 5.5, page 18) so no further

revision was deemed necessary.

24. Concerning the comment on the relevance of the Beier et al. (2008) study on triclosanresistance, the SCCS is of the view that the study investigated the susceptibility profile of

vancomycin-resistant Enterococcus faecium isolates from community wastewater. One third of

the isolates showed MICs of up to 8 µg/ml. This confirms that there was no correlation betweenantibiotic susceptibility profile and biocide susceptibility. In the view of the SCCS the study

showed a specific mechanism of high resistance to vancomycin and is not correlated to any

known mechanism of biocide resistance.

25. Concerning the comment on the possibility to predict changes in antibiotic resistance of

bacteria following exposure to triclosan, the SCCS agrees with the view of the SCENIHR

(2010) that, on the basis of the available evidence in the scientific literature, it is not possible at

present to predict changes in the antibiotic resistance profiles of bacteria following exposure to

triclosan or to any other of the biocides currently used in various applications.




SCCP/1251/09

Scientific Committee on Consumer Safety

SCCS


Antimicrobial Resistance

The SCCS approved this opinion at its 7th plenary of 22 June 2010 after public consultation



SCCP/1251/09

Opinion on triclosan – antimicrobial resistance

About the Scientific CommitteesThree independent non-food Scientific Committees provide the Commission with thescientific advice it needs when preparing policy and proposals relating to consumer safety,

public health and the environment. The Committees also draw the Commission's attentionto the new or emerging problems which may pose an actual or potential threat.They are: the Scientific Committee on Consumer Safety (SCCS), the Scientific Committeeon Health and Environmental Risks (SCHER) and the Scientific Committee on Emerging andNewly Identified Health Risks (SCENIHR) and are made up of external experts.

In addition, the Commission relies upon the work of the European Food Safety Authority(EFSA), the European Medicines Evaluation Agency (EMEA), the European Centre forDisease prevention and Control (ECDC) and the European Chemicals Agency (ECHA).

SCCSThe Committee shall provide opinions on questions concerning all types of health and safetyrisks (notably chemical, biological, mechanical and other physical risks) of non-food

consumer products (for example: cosmetic products and their ingredients, toys, textiles,clothing, personal care and household products such as detergents, etc.) and services (forexample: tattooing, artificial sun tanning, etc.).

Scientific Committee membersJürgen Angerer, Ulrike Bernauer, Claire Chambers, Qasim Chaudhry, Gisela Degen, GerhardEisenbrand, Thomas Platzek, Suresh Chandra Rastogi, Vera Rogiers, Christophe Rousselle,Tore Sanner, Kai Savolainen, Jacqueline Van Engelen, Maria Pilar Vinardell, RosemaryWaring, Ian R. White

ContactEuropean CommissionHealth & ConsumersDirectorate C: Public Health and Risk AssessmentUnit C7 - Risk AssessmentOffice: B232 B-1049 [email protected]

© European Union, 2010

ISSN 1831-4767 ISBN 978-92-79-12484-6

Doi:10.2772/11162 ND-AQ-09-001-EN-N

The opinions of the Scientific Committees present the views of the independent scientistswho are members of the committees. They do not necessarily reflect the views of theEuropean Commission. The opinions are published by the European Commission in theiroriginal language only.

http://ec.europa.eu/health/scientific_committees/consumer_safety/index_en.htm

mailto:[email protected]









SCCP/1251/09


ACKNOWLEDGMENTS

Dr. J. Davison, Retired, Member of SCHER, FRDr. J.-Y. Maillard, Cardiff University, UKDr. J.-M. Pagès, University of Marseille, Member of SCENIHR, FR (Rapporteur)Dr K. Pfaff, BfR, DEDr. S.C. Rastogi, Retired, Member of SCCS, DK (Chairman)

Keywords: SCCS, scientific opinion, preservative, triclosan, P32, antimicrobial resistancedirective 76/768/ECC, CAS 3380-34-5, EC 222-182-2

Opinion to be cited as: SCCS (Scientific Committee on Consumer Safety), Opinion ontriclosan (antimicrobial resistance), 22 June 2010



SCCP/1251/09


Table of Content

ACKNOWLEDGMENTS...........................................................................3

ABSTRACT..........................................................................................6

EXECUTIVE SUMMARY..........................................................................6

1. BACKGROUND..............................................................................8

2. TERMS OF REFERENCE..................................................................8

3. INTRODUCTION ...........................................................................9

3.1. Scope.............................................. ........................................................... ........................................................ 9

3.2. Physico-chemical properties.......... ...................................................................... ......................................... 10

3.3. Triclosan in biocidal formulations.............................................................. ................................................. 12

3.4. Mode of action ........................................................... ............................................................... ..................... 13

4. DEFINITIONS.............................................................................13

5. PRODUCTION, USE AND FATE OF TRICLOSAN.................................15

5.1. Triclosan in cosmetics .......................................................... ............................................................. ............ 15

5.2. Triclosan in healthcare and medical devices............................................................................................... 16

5.3. Triclosan in household and other consumer products .................................................................... ........... 16

5.4. Triclosan in food and feed ............................................................... .......................................................... ... 17

5.4.1. Triclosan in food production............................................................... ..................................................... 17

5.4.2. Triclosan as disinfectant in food and feed production ............................................................... ............ 17

5.4.3. Triclosan as food preservative .......................................................... ....................................................... 17

5.4.4. Triclosan in animal husbandry ........................................................... ..................................................... 18

5.4.5. Triclosan as feed preservative................................................................ .................................................. 18

5.5. Triclosan in the environment ......................................................... ............................................................ .. 18

5.5.1. Fate of triclosan in the environment...................................................................... .................................. 18

5.5.2. Effect of triclosan on micro-flora and toxicity of metabolites ............................................................... 21

5.6. Triclosan in the human body................................................................... ..................................................... 22



SCCP/1251/09


6. MECHANISMS OF RESISTANCE TO TRICLOSAN............................... 22

6.1. General considerations on biocide resistance in bacteria.................................................................. ........ 22

6.2. General considerations on the study of triclosan............................................................................. ........... 24

6.3. Mechanisms of bacterial resistance to triclosan ................................................................... ...................... 24

6.4. Mutation rates and transfer of resistance ............................................................... .................................... 27

6.5. Induction of resistance........... ................................................................ ....................................................... 27

6.6. Bacterial cross-resistance to triclosan and antibiotics .................................................................... ........... 28

6.6.1. General considerations .......................................................... ................................................................. .. 28

6.6.2. Triclosan and cross-resistance .......................................................... ....................................................... 28

6.7. Triclosan resistance in bacteria in situ ..................................................... ................................................... 30

7. TRICLOSAN BIOAVAILABILITY AND FORMULATION EFFECTS.............31

8. MEASUREMENT OF RESISTANCE AND CROSS-RESISTANCE .............. 32

9. DATA GAPS ON SCIENTIFIC KNOWLEDGE......................................33

9.1. Scientific gaps:............................................................ .............................................................. ..................... 33

9.2. Technical gaps: ............................................................ ............................................................. ..................... 33

10. RISK ASSESSMENT................................................................... 33

10.1. Limitation in activity................................................. ........................................................... ..................... 34

10.2. Genetic and bacterial point of view .................................................................... ..................................... 34

10.3. Environment point of view ................................................................ ....................................................... 34

10.4. Biofilm formation in specific environmental conditions...... ................................................................ .. 35

11. CONCLUSIONS......................................................................... 35

12. OPINION................................................................................. 36

13. COMMENTS RECEIVED DURING THE PUBLIC CONSULTATION.........37

14. MINORITY OPINION.................................................................. 37

15. REFERENCES........................................................................... 38



SCCP/1251/09


ABSTRACT

Triclosan is a biocide used in many product categories, including cosmetics. The information

on environmental concentrations of triclosan in the EU is limited and the bioavailability ofthe triclosan to bacteria in the environment is not known.

Although the present mandate concerns the evaluation of a possible association betweenthe use of triclosan in cosmetic products and the development of resistance by certainmicro-organisms, the SCCS has taken into account all evidence available from all uses oftriclosan to perform its assessment.

A number of scientific and technical data gaps about the occurrence and understanding ofthe resistance profile of triclosan have been identified and should be addressed.

At present, several distinct hazards have been identified: (i) the effect of triclosan on thetriggering/regulation of resistance genes in bacteria (ii) the existence of definedmechanisms that can promote resistance and cross-resistance to biocides and antibiotics in

bacteria, (iii) high concentrations of triclosan (compared to concentrations known to selectfor resistance in in vitro experiments) have been measured in certain environmentalcompartments and (iv) bacterial biofilms, which are widespread in the environment and areable to survive exposure to adverse environmental factors. The first two of these hazardshave been identified in vitro. The presence of resistance genes in soil bacteria should beinvestigated further.

Based on the six in situ studies and the one meta-analysis quoted in this document andrecent data from in vitro investigations (proteomic and genomic analyses), it is not possibleto quantify the risk associated with triclosan (including its use in cosmetics) in terms ofdevelopment of antimicrobial resistance (i.e. selection for less susceptible population),genetic basis for resistance and dessemination of resistance. In view of the concentrationsof triclosan reported to trigger resistance in vitro, some of the environmental concentrations

found in a number of geographical distinct areas are high enough to suggest that bacterialresistance could be triggered. However, no studies have been conducted on this aspect. Theapplications of triclosan which contribute to those high environmental concentrations cannotbe properly identified nor quantified at present and the presence of other chemicals (e.g.antibiotics, surfactants, other biocides, etc.) in the environment, which may also affectmicrobial populations, would preclude assessing the effects of triclosan independently. Thus,additional in situ information is needed to provide an answer on the level of risk.

EXECUTIVE SUMMARY

Triclosan is a biocide used in many product categories, including cosmetics. The informationon environmental concentrations of triclosan in the EU is limited and bioavailability of thetriclosan to bacteria in the environment is not known.

Although the present mandate concerns the evaluation of a possible association betweenthe use of triclosan in cosmetic products and the development of resistance by certainmicro-organisms, the SCCS has taken into account all evidence available from all uses oftriclosan to perform its assessment.

Triclosan is the most studied biocide with respect to bacterial resistance. Such a level ofinformation, notably on its activity against bacteria, the identification of mechanisms ofmicrobial resistance including genomic and proteomic aspects, is commendable and shouldbe extended to other biocides.

Low concentrations of triclosan can trigger the expression of resistance and cross-resistancemechanisms in bacteria in vitro. In view of the concentrations of triclosan reported to



SCCP/1251/09


geographical distinct areas are high enough to suggest that bacterial resistance could betriggered. It is however difficult to predict whether microbial resistance would be triggeredin these environments. The few in situ studies performed to date did not show any bacterialresistance emerging following triclosan exposure. In addition, the presence of otherchemicals (e.g. antibiotics, surfactants, other biocides, etc.) in the environment, which may

also affect microbial populations, would preclude assessing the effects of triclosanindependently.

The emergence of resistance induced/selected by triclosan is related to the genetic controlon the resistance gene(s) present on chromosomal and genetic mobile elements. Thisrepresents the origin for a hazard about selection and dissemination of cross-resistance withother anti-bacterial molecules including biocides and antibiotics.

Triclosan, like any other biocide, contributes to the selection of less susceptible bacteria in acomplex microcosm in vitro. The impact of such a selection is unclear, as is the fitness ofthe “selected” bacterial species following triclosan exposure. The few in situ studiesinvestigating long-term triclosan exposure (i.e. at least 6 months) did not indicate changesin the resistance susceptibility in the predominant bacteria selected for monitoring, but the

changes in the entire flora were not evaluated. Thus additional in situ information is neededto provide a definitive opinion.

There are, so far, no epidemiological data linking outbreaks of antimicrobial resistant humanand zoonotic pathogens to exposure to triclosan.

A number of scientific and technical data gaps about the occurrence and understanding ofthe resistance profile of triclosan have been identified and should be addressed. Inparticular, where biocides, including triclosan are used intensely, monitoring for emergingresistance in the microbial flora should be conducted. A more detailed research strategy forinvestigating the antimicrobial resistance effect of biocides is presented in a separateopinion from the SCENIHR (2010).

There is an apparent discrepancy between in situ information that suggests the absence ofinduction of bacterial resistance and cross-resistance triggered by triclosan, and in vitro studies describing the mechanistic and genetic aspect of triclosan-resistance in bacteria. Abetter translation of in vitro findings to in situ situations is needed, making full use ofmolecular tools and environmental conditions used in laboratory investigations.Standardized protocols and similar parameters should be applied to both in vitro and in situ investigations.

Although triclosan resistance was not observed in situ, this is not sufficient to conclude thatthere is no risk. Information is still lacking to provide a risk assessment on the use oftriclosan in cosmetic products, including the genetic aspects of resistance, changes inenvironmental microcosm, maintenance and transfer of virulence and resistancedeterminants in situ.

Due to the limited number of in si tu studies of resistance induced by triclosan to date, SCCScan only recommend the prudent use of triclosan, for example in applications where ahealth benefit can be demonstrated. However, conclusions from in vitro studies cannot beignored, notably the role of triclosan (and other biocides) in triggering resistance and in thedissemination (horizontal or vertical transfer of) resistance determinants. Research focusedon triggering mechanisms of resistance, maintenance of the gene pool and the transfer ofresistance and virulence determinants, and improving the translational application oflaboratory results to situations in situ are needed. Hence,the SCCS appreciates thatresearch investment from the industry will be maintained to contribute to a betterunderstanding of the potential risks associated with triclosan applications.



SCCP/1251/09


1. BACKGROUND

Triclosan (CAS 3380-34-5) with the chemical name 5-chloro-2-(2,4-dichlorophenoxy)phenol

or 2,4,4'-trichloro-2'-hydroxy-diphenyl ether has a long history of use as a preservative incosmetic products. It is currently regulated in Annex VI, entry 25 with a maximumconcentration of 0.3%.

An opinion on triclosan (SCCP/1040/06) was adopted by the SCCP at the 9 th plenarymeeting of 10 October 2006 with the following conclusions to the request:

1. "On the basis of the available data, the SCCP is of the opinion that there is presently

no evidence of clinical resistance and cross-resistance occurring from the use oftriclosan in cosmetic products. Information is required on consumer exposure totriclosan from all sources, including cosmetic products.

2. For a toxicological assessment of the safe use of triclosan, the SCCP requires a dossierto be submitted in which data is provided to all relevant exposure and toxicologicalend-points and conforming to currently accepted standards. This should be regarded

as a matter of urgency because triclosan has been identified in human milk of someEuropean populations."

The dossier provided by Industry consists of an update on the bacterial resistance issue(submission III) and of a toxicological dossier for triclosan (submission IV).

Furthermore the Norwegian authority on cosmetics has earlier this year submitted a report"Risk assessment on the use of triclosan in cosmetics; Development of antimicrobialresistance in bacteria – II".

2. TERMS OF REFERENCE

Does SCCS consider a continued use of triclosan as a preservative in cosmetic products as safe taking into account the new provided documentation of resistancedevelopment by certain micro-organisms and cross-resistance?

In parallel, the SCCP/SCCS has been asked to assess the toxicological safety of triclosanwhen used as a preservative with a maximum concentration of 0.3%. This evaluation has

been published as opinion SCCP/1192/08.



SCCP/1251/09


3. INTRODUCTION

Triclosan is an antimicrobial agent that has been used for more than 40 years as anantiseptic, disinfectant or preservative in clinical settings, in various consumer products

including cosmetics, plastic materials, toys, etc. It has a broad range of activity thatencompasses many, but not all, types of Gram-positive and Gram-negative non-sporulating,bacteria, some fungi (Jones et al . 2000, Schweizer 2001), Plasmodium falciparum andToxoplasma gondii (McLeod et al . 2001). It has also been shown to be ecotoxic, particularlyto algae in aquatic environments (Tatarazako et al . 2004). Additionally, it has been shownto interfere with the cycling of nitrogen in natural systems (Fernandes et al . 2008, Wallerand Kookana 2009).

Triclosan is bacteriostatic at low concentrations, but higher levels are bactericidal (Sullerand Russell 1999, 2000). At sublethal concentrations, it acts by inhibiting the activity of thebacterial enoyl-acyl carrier protein reductase (FabI), a critical enzyme in bacterial fatty acidbiosynthesis (Heath et al . 2002, Zhang et al . 2004). At bactericidal concentrations, it issuggested to act through multiple nonspecific mechanisms including membrane damage

(Gilbert and McBain 2002).

There are concerns that the widespread use of a low concentration of triclosan in variousapplications might lead to or select for bacterial resistance to antibiotics. Antibioticresistance has become an increasingly serious problem worldwide, and the continued use ofbiocides including triclosan may exacerbate this problem. The main cause of antibioticresistance remains the use and misuse of antibiotics. During the last decade, antibioticresistance has increased in bacterial pathogens leading to treatment failures in both humanand animal infectious diseases (Harbarth and Samore 2005; for reports see: EARSS AnnualReport 2005, WHO 2007).

The safety of continued use of triclosan in cosmetic products has recently been assessed bythe EU Scientific Committee on Consumer Products (SCCP 2009). The SCCP emphasised

that this risk assessment concerns only the toxicological profile of triclosan and that beforea final conclusion on the safety of triclosan in cosmetic products can be reached, thepotential development of resistance to triclosan and cross-resistance by certain micro-organisms must be assessed. Earlier evaluations of triclosan, on the basis of available data,EU Scientific Committees concluded that there was no convincing evidence that triclosanposes a risk to humans and environment by inducing or transmitting antibacterial resistance(SSC 2002) as well as there was no evidence of clinical resistance and cross-resistanceoccurring from the use of triclosan in cosmetic products (SCCP 2006). Further informationwas sought for an update of these evaluations.

The present evaluation of triclosan is based both on the information submitted by COLIPA1 to SCCS and on research published in peer-reviewed scientific journals. It aims atdetermining whether the continued use of triclosan may be associated to the development

of resistance in certain micro-organisms. It also aims at identifying additional researchneeds.

3.1. Scope

Triclosan is used as a preservative in consumer products including cosmetics, where themaximum allowed concentration according to the EU Cosmetics Directive 76/768/EEC is0.3%. The SCCP has recently performed a risk assessment of the use of triclosan incosmetic products. Although the present mandate concerns the evaluation of a possibleassociation between the use of triclosan in cosmetic products and the development ofresistance by certain micro-organisms, the SCCS has taken into account all evidence

available from all uses of triclosan to perform its assessment. This is in line with the SCCP



SCCP/1251/09


conclusions of 2006 (SCCP/1040/06) and it is scientifically sound as 1) cosmetic uses oftriclosan account for most of the total use of this biocide in the EU and 2) it is scientificallyimpossible at present to assess the use of triclosan in cosmetics only, without taking intoaccount its uses in other applications. In the absence of a clear answer, research needs willbe identified. The effect of triclosan on microflora in the environment on the basis of

published literature will also be covered, since environmental bacteria represent a pool ofantimicrobial resistance genes.

Most of the information provided here relates to bacteria, since studies of the effects oftriclosan on other micro-organisms are scarce.

3.2. Physico-chemical properties

INCI Name: Triclosan

Chemical Name: 2,4,4’-trichloro-2’-hydroxy-diphenylether

Synonyms: Phenol, 5-chloro-2-(2,4-dichlorophenoxy)-; Ether, 2'-hydroxy-2,4,4'-

trichlorodiphenyl; 5-Chloro-2-(2,4-dichlorophenoxy)phenol, Trichloro-2'-

hydroxydiphenylether

Trade Names: Irgasan® DP300, Irgasan® PG60, Irgacare® MP, Irgacare® CF100,

Irgacide® LP10, ; Cloxifenolum, Irgagard® B 1000, Lexol 300, Ster-Zac

CAS Reg. No.: 3380-34-5

EC: 222-182-2

Chemical structure:

O

CI

OHCI

CI

Empirical formula: C12H7Cl3O2

Molecular weight: 289.5Physical form: White crystalline powder

The purity of batches of triclosan used in personal care products since the 1970s isdescribed in the Table 1 (SCCP 2009). These data were provided by COLIPA. The purity andcontaminants might be different in triclosan from other sources.



SCCP/1251/09


Table 1: Purity specifications for triclosan since 1970

TestPoint

Effectivefrom

1970

Effectivefrom

26.09.1985

Effectivefrom

1.1.1990

Effectivefrom31.12.1994



TriclosanActiveSubstance1

99.0 -100.0%

99% min 99% min 99.0-100% 97.0-103.0%

97.0 -103.0%

1 Analysis by gas chromatography.

Impurities / accompanying contaminants: See Table 2.

Table 2: Impurities in triclosan

Individual related compound (Gas Chromatography) ≤0.1%Total related compounds (Gas Chromatography) ≤ 0.5%

2,4 Dichlorophenol ≤10 mg/kg

Sum of 3- and 4-Chlorophenol ≤10 mg/kg

2,3,7,8 Tetrachlorodibenzo-p-dioxin <0.001 µg/kg

2,3,7,8-Tetrachlorodibenzo-furan <0.001 µg/kg

2,8-Dichlorodibenzo-p-dioxin ≤0.5 mg/kg

1,3,7-Trichlorodibenzo-p-dioxin ≤0.25 mg/kg

2,8-Dichlorodibenzo-furan ≤0.25 mg/kg

2,4,8-Trichlorodibenzo-furan ≤0.5 mg/kg

Ash ≤0.1%

Mercury ≤1 mg/kg

Arsenic ≤2 mg/kg

Antimony 10 mg/kg

Lead ≤10 mg/kg

Cadmium ≤5 mg/kg

Nickel ≤10 mg/kg

Copper ≤10 mg/kg

Chromium ≤2 mg/kg

Sum of heavy metals as lead sulfide precipitation ≤20 mg/kg

Partition coefficient: Log Pow = 4.8

Melting point: 57 ± 1°C

Relative density: 1.55 ± 0.04 g/cm3

Vapour pressure: 4 x 10-6 mmHg (20°C)

pKa: 8.14 (20°C)

Stability: Triclosan does not decompose under normal storage conditions over 9 years ofstorage (information from COLIPA).



SCCP/1251/09


The solubility of triclosan is described in Table 3.

Table 3: Solubility of triclosan in selected solvents and chemicals

Solvent Solubility at 25°C(g Triclosan/100 g solvent)



1 N caustic soda 31.7

1 N sodium carbonate 0.40

1 N ammonium hydroxide 0.30

Triethanolamine >100

Acetone >100

Ethanol 70% or 95% >100

Isopropanol >100

Propylene glycol >100

Polyethylene glycol >100

Methyl cellosolve (Union CarbideCorp.)

>100

Ethyl cellosolve (Union CarbideCorp.)

>100

Dipropylene glycol ~40Glycerine 0.15

n-Hexane 8.5

Petroleum jelly (white, USP) ~0.5



Triton X-100 (Rohm & Haas) >100

Olive oil ~60

Castor oil ~90

3.3. Triclosan in biocidal formulations

Biocidal products that contain triclosan as the main antimicrobial are usually complexformulations due to the lack of solubility of this bisphenol. Components of the formulationmight affect the activity of triclosan positively (e.g. through synergism) or negatively (e.g.antagonism). There is some information on the effect of formulation components on biocideactivity (Alakomi et al . 2006, Ayres et al . 1999, Denyer and Maillard 2002, Maillard 2005b),but by large this information is restricted due to proprietory restrictions, or the lack ofunderstanding on how formulation components work in term of antimicrobial potentiation.

In the scientific literature, where triclosan acitivity has been reported, there is littlereference to the use of formulation. Instead triclosan is often dissolved in a solvent such asDMSO.



SCCP/1251/09


3.4. Mode of action

Chemical biocides are generally considered to have multiple target sites against microbialcells, although such interactions are concentration dependent (Russell et al . 1997; Maillard2005a). The bisphenol triclosan is no exception. At a sub-inhibitory concentration, triclosan

was found to profoundly affect bacterial growth, indicating a strong interaction with thebacterial targets, despite the high concentration exponent of triclosan (McDonnell andRussell 1999). At higher concentrations, Gomez Escalada et al . (2005a) observed thattriclosan was both rapid-acting and active at all phases of population growth, although somemarked differences in its lethality were observed.

These observations substantiated earlier findings with Staphylococcus aureus (Regos andHitz 1974; Suller and Russell 2000). Inhibition of key metabolic pathway and synthesis(Regos and Hitz 1974; McMurry et al . 1998b) might be part of the lethal action mechanismsof triclosan. Indeed, triclosan was found to target specifically fatty acid synthesis with theinhibition of the enzyme enoyl reductase (enoyl-acyl carrier protein reductase, FabI)(McMurry et al . 1998a). It acts as a potent irreversible inhibitor of FabI by mimicking itsnatural substrate (Heath et al . 1998; Levy et al . 1999) and this inhibition has been

described as being slow and competitive (Heath et al . 1999). The propensity of triclosan toinhibit fatty acid synthesis in Plasmodium falciparum and Toxoplasma gondii (McLeod et al .2001) has led to the development of a number of antimalarial and antibacterial pro-drugsbased on triclosan (Mishra et al . 2008; Freundlich et al . 2009).

The rapid action of triclosan at a high concentration might be indicative of membranedamage (Villalain et al . 2001) and it is clear that fatty acid synthesis targeting cannot solelyexplain the lethal effect of triclosan (Gomez Escalada et al . 2005b). Triclosanmembranotropic effects result in destabilised structures compromising the functionalintegrity of cell membranes without inducing cell lysis (Villalain et al . 2001). Intercalation oftriclosan into bacterial cell membranes is likely to compromise the functional integrity ofthose membranes, thereby accounting for some of triclosan antibacterial effects (Guillén etal . 2004).

Recently, the first genome-wide transcriptional analysis of Staphylococcus aureus exposedto triclosan (0.05 µM), reported that triclosan down regulated primary metabolism-relatedand carbohydrate transport, the cap operon which is essential for virulence, the clpB chaperone-related genes which might trigger the expression of resistant determinants,genes involved in fatty acid production and utilisation (Jang et al . 2008).

A number of factors affect the antimicrobial activity of triclosan. These can be divided intointrinsic factors derived from the biocide and its application (e.g. concentration, contacttime, pH) and extrinsic factors which derive from the environment during application (e.g.temperature, soiling). Understanding the complex relationship between concentration andcontact time (sometimes referred to as CT concept) is crucial to ensure efficacy (Maillard2005a). The stability of triclosan in particular environments will also influence efficacy.

4. DEFINITIONS

According to the Directive 98/8/EC of the European Parliament and Council of the 16February 1998, biocidal products are defined as active substances and preparationscontaining one or more active substances, put up in the form in which they are supplied tothe user, intended to destroy, render harmless, prevent the action of, or otherwise exert acontrolling effect on any harmful organism by chemical or biological means.

Within the scope of this mandate, the proposition is to apply the following definitions:

• Antimicrobial: biocide or antibiotic.



SCCP/1251/09


• Biocide: an active chemical molecule in a biocidal product to control the growth of orkill micro-organisms (including bacteria, fungi, protozoa and viruses). This includesdisinfectants, preservatives and antiseptics.

• Antibiotic: an active substance of synthetic or natural origin which is used to

eradicate bacterial infections in humans or animals.• Antimicrobial activity: an inhibitory or lethal effect of a biocidal product or an

antibiotic.

The terms employed in the context of this mandate are defined below in order to avoidconfusion in the definitions used to describe the level and type of resistance reported.

There are several definitions of resistance to antimicrobials biocides or/and antibiotics andseveral terms used to describe similar phenomena in the literature. A literal/biologicaldefinition of resistance is the capacity of bacteria to withstand the effects of a harmfulchemical agent.

The following definitions are based partly on those put forward by Chapman and colleagues

(Chapman 1998, Chapman et al . 1998), Russell and colleagues (Hammond et al . 1987,Russell 2003) and Cloete (2003), and the recent SCENIHR opinion (2009).

The practical meaning of antibiotic resistance is to describe situations where (i) a strain isnot killed or inhibited by a concentration attained in vivo, (ii) a strain is not killed orinhibited by a concentration to which the majority of strains of that organism aresusceptible or (iii) bacterial cells that are not killed or inhibited by a concentration actingupon the majority of cells in that culture.

When non-antibiotic agents (i.e. triclosan or other biocides) are considered, the word “resistance” is used in a similar way where a strain is not killed or inhibited by aconcentration attained in practice (the in-use concentration) and in situations (ii) and (iii)described above.

These definitions reflect those given by EFSA whereby “antimicrobial susceptibility orresistance is generally defined on the basis of in vitro parameters. The terms reflect thecapacity of bacteria to survive exposure to a defined concentration of an antimicrobialagent, but different definitions are used depending on whether the objective of theinvestigation is clinical diagnostics or epidemiological surveillance” (EFSA 2008)

The term 'Multi-Drug Resistant’ (MDR) applies to a bacterium that is simultaneouslyresistant to a number of antibiotics belonging to different chemical classes by using variousmechanisms (Depardieu et al . 2007).

The term “co-resistant” is used to denote a strain possessing a biochemical mechanism thatinhibits the activity of several antibiotics belonging to the same structural family (e.g. ß-lactamase and ß-lactams). When the transfer of resistance determinants occurs, co-

resistance specifically refers to genetic determinants (such as integrons, transposons orplasmids) encoding for unrelated resistance mechanisms, that are transferred in a singleevent and expressed jointly in a new bacterial host.

The term “cross-resistant” is used to denote a strain possessing a resistance mechanismthat enables it to survive the effects of several antimicrobial molecules with mechanism(s)of action that are related or overlap.

Other terms such as “insusceptibility” and “tolerance” have been used in the publishedliterature. Insusceptibility refers to an intrinsic (innate) property of a micro-organism, suchas cell layer impermeability in mycobacteria and Gram-negative bacteria. Tolerance denotesa reduced susceptibility to an antimicrobial molecule characterised by a raised minimuminhibitory concentration (MIC), or a situation in which a preservative system no longerprevents microbial growth.



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5. PRODUCTION, USE AND FATE OF TRICLOSAN

Triclosan is a broad spectrum antimicrobial used as an antiseptic, disinfectant orpreservative in clinical settings, various consumer products including cosmetics, householdcleaning products, plastic materials, toys, paints, etc. It is also incorporated on the surface

of medical devices, plastic materials, textiles, kitchen utensils, etc., from which it mightslowly leach for a long period of time during their use, to perform its biocidal action. Adetailed list of products containing triclosan is provided by the US Environmental ProtectionAgency (EPA) (McMahon et al . 2008) and by the Environmental Working Group, a US NGO(http://www.ewg.org/node/26752). According to EU Biocide Directive 1998/8/EC, triclosanis used in product types 1 (human hygiene), 2 (private and public health area), 3(veterinary hygiene), 7 (film preservative) and 9 (fibre, leather, rubber and polymerisedmaterials preservative).

According to the information provided by COLIPA, the quantity of triclosan used within theEU reached approximately 450 tons (as 100% active) in the year 2006. Dye et al . (2007)estimated triclosan production in the EU to be 10-1,000 tonnes per year. It is not clearwhether the above information on use or production of triclosan includes the amounts of

triclosan which may be imported in the EU or exported from the EU via finished products,such as medical devices, toys, textiles, etc. In the EU, about 85% of the total volume oftriclosan is used in personal care products, compared to 5% for textiles and 10% for plasticsand food contact materials (usage data reported by COLIPA in 2007).

The Danish EPA performed a survey of the use of triclosan in Denmark for the period 2000-2005 (Borling et al . 2005). This survey showed that the amount of triclosan in products onthe Danish market had decreased from approx. 3.9 to 1.8 tonnes (54%) in the period 2000-2004. Cosmetics were the largest contributor to the amount of triclosan on the Danishmarket (99% of the total reported amount in the survey). However, this might not berespresentative for the whole EU, as similar data for comparison is not available for the EUas a whole or for any of its Member States.

5.1. Triclosan in cosmetics

Triclosan was listed in 1986 in the European Community Cosmetics Directive (76/768/EEC)for use as a preservative in cosmetic products at concentrations up to 0.3%. The recent riskassessment performed by the EU Scientific Committee on Consumer Products (SCCP)concluded that, although its use at a maximum concentration of 0.3% in toothpastes, handsoaps, body soaps/shower gels and deodorant sticks was considered safe on a toxicologicalpoint of view in individual products, the magnitude of the aggregate exposure to triclosanfrom all cosmetic products is not safe. Any additional use of triclosan in face powders andblemish concealers at this concentration was also considered safe, but the use of triclosan inother leave-on products (e.g. body lotions) and in mouthwashes was not considered safe for

the consumer due to the resulting high exposures2. Inhalation exposure to triclosan fromspray products (e.g. deodorants) was not assessed.

In a Danish EPA survey (Borling et al . 2005), the highest amount of triclosan in cosmeticswas found in products for dental hygiene, including toothpaste. In this group, the amounthad decreased by 37% during 2000-2004. Deodorants were the group of cosmetics with thegreatest decrease in amount of triclosan (79%). A recent Danish EPA survey revealed that15% of the most commonly sold deodorants in the Danish market contained <0.3%triclosan (Rastogi et al . 2007).

Triclosan being non-ionic, it can be formulated in conventional dentifrices. However, it doesnot bind to the oral surfaces for more than a few hours, and therefore does not deliver asustained level of anti-plaque activity. To increase uptake and retention of triclosan by oral

2 SCCP opinion on triclosan COLIPA n° P32 SCCP/1192/08

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surfaces for the improvement of plaque control and gingival health,triclosan/polyvinylmethyl ether maleic acid copolymer and triclosan/zinc citrate andtriclosan/calcium carbonate dentifrice are used (Williams 1998, Davies et al . 2004, Bradinget al . 2004, Davies 2007).

5.2. Triclosan in healthcare and medical devices

Triclosan has been effectively used clinically to eradicate micro-organisms such asmethicillin-resistant Staphylococcus aureus (MRSA) (Brady et al . 1990; Cookson et al .1991; Webster et al . 1994; Zafar et al . 1995), notably with the recommendation to use 2%triclosan bath. Triclosan is employed as surgical scrubs, and it is widely used in handwashing (Boyce and Pittet 2002) and as a body wash to eradicate MRSA from carriers priorto surgery (Wilcox et al . 2003).

Triclosan is used in a number of medical devices, for example ureteral stents (Knudsen et

al . 2005), surgical sutures (Ford et al . 2005; Justinger et al . 2009) and might be consideredto prevent graft infection (Cakmak et al. 2009). Bojar et al. (2009) did not observe a

difference in colonisation between triclosan-coated sutures and regular multifilament suture,although their work concerned five bacteria and is only based on the determination of thezone of inhibition. In ureteral stents, triclosan has been shown to inhibit the growth ofcommon bacterial uropathogens and to reduce the incidence of urinary-tract infections and,potentially, catheter encrustation (Chew et al . 2006, Cadieux et al . 2009). Wignall et al .(2008) have recently demonstrated synergistic effects of triclosan and relevant antibioticson clinical isolates comprising seven uropathogenic species, and they support the use of thetriclosan-eluting stent when necessary, along with standard antibiotic therapy in treatingcomplicated patients. In some further developments, the use of triclosan in urinary Foleycatheter was suggested since triclosan successfully inhibited the growth of Proteus mirabilis and controlled encrustation and blockage of the catheter (Stickler et al . 2003, Williams andStickler 2008). Recently, Darouiche et al . (2009) demonstrated synergistic, broad-spectrum

and durable antimicrobial activity of the catheters coated with a combination of triclosanand DispersinB, an anti-biofilm enzyme that inhibits and disperses biofilms (Kaplan et al .2004, Itoh et al . 2005).

5.3. Triclosan in household and other consumer products

The broad-spectrum antimicrobial activity of triclosan has led to its incorporation in anextended range of product formulations intended for home use such as liquid soaps,detergents, chopping boards, children’s toys, carpets and food storage containers (Bhargavaand Leonard 1996, McBain et al . 2003, Yazdankhah et al . 2006, Gilbert et al . 2007). Adetailed list of consumer products containing triclosan is provided by the US EnvironmentalProtection Agency (EPA) (McMahon et al . 2008) and by the US NGOs "Environmental

Working Group" (http://www.ewg.org/node/26752) and "Beyond Pesticides"(http://www.beyondpesticides.org/antibacterial/products.htm).

An increasing number of clothing articles are treated with biocides. Triclosan is one of thefinishing agents for the production of such textiles (Orhan et al . 2009).The fabrics finishedwith triclosan are treated with cross-linking agents to provide durable antibacterialproperties. On the basis of the available information, 17 products from the Danish retailmarket were analysed for the content of some selected antibacterial compounds: triclosan,dichlorophen, Kathon 893, hexachlorophen, triclocarban and Kathon CG. Five of theproducts were found to contain 0.0007% - 0.0195% triclosan (Rastogi et al . 2003).

Aiello et al . (2007), in the first systematic review assessing the benefit of soaps containingtriclosan, evaluated 27 studies published between 1980 and 2006. One of the key findings isthat soaps that contained less than 1% triclosan showed no benefit from non-antimicrobialsoaps (the EU limit is 0.3%). Studies that used soap contaning > 1% triclosan showed asignificant reduction in bacterial levels on hand, often after multiple applications. Theapparent lack of relationship between the use of soap containing triclosan and reduction in




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infectious illness was difficult to ascertain in the absence of identification of the biologicalagents responsible for the illness symptoms. Two recent US studies (Fischler et al . 2007,Fuls et al . 2008) demonstrated that hand washing with antimicrobial soap containingtriclosan (0.46%) reduced bacterial load and transfer of bacteria from hands, compared tohandwashing with a non-antimicrobial soap.

5.4. Triclosan in food and feed

5.4.1. Triclosan in food production

Triclosan was evaluated by the Scientific Committee on Food (SCF 2000) and the EuropeanFood Safety Authority (EFSA 2004) for use in food contact materials and classified in SCFList 33 with a restriction of 5 mg/kg of food. The evaluation was referred to the use oftriclosan as surface biocide i.e. as substance intended to inhibit the growth of bacteria on

the surface but which is not intended to have an antimicrobial effect on the food itself.Potential uses beyond household articles like cutting boards, kitchen utensils and foodstorage containers exist (e.g. conveyor belts, machinery, work surfaces and transportcontainers used in food processing). However, in April 2009 the petitioner has withdrawnthe application for these uses. According to a March 2010 Commission Decision4 triclosanshall not be included in the positive list of additives to Directive 2002/72/EC and cannot beused in the manufacture of plastics intended to come into contact with food.

In Germany, the use of triclosan in food contact plastics is banned since September 2009.BfR supports the ban on triclosan in food contact materials (BfR Opinion N°. 031/2009, 12June 2009).

Triclosan has been identified in drinking water in certain places (Stackelberg et al . 2004,Boyd et al . 2003). Kantiani et al . (2008) found methyl triclosan (12 µg/L) in one of the 22

drinking water samples from Barcelona.

5.4.2. Triclosan as disinfectant in food and feed production

Triclosan is not notified in the framework of the European regulations on biocides (Directive98/8/EC) for use as disinfectant in food and feed production.

5.4.3. Triclosan as food preservative

Triclosan is not approved as food preservative in Europe. Food preservatives are regulatedby Directive 95/2/EC on food additives other than colours and sweeteners. In Annex III of

this Directive on the permitted preservatives and restrictions for their use, triclosan is notlisted. As a result, the use of triclosan in so-called “active food contact materials andarticles” is not allowed. Regarding substances released from such materials in order toextend the shelf-life of food, the Regulation (EC) 1935/2004 on food contact materialsrefers to the authorisations applicable to their use in foods.

3 Substances for which an Acceptable Daily Intake or Tolerable Daily Intake could not be established, but where thepresent use could be accepted.

4 Commission Decision of 19 March 2010 concerning the non-inclusion of 2,4,4’-trichloro-2’-hydroxydiphenyl ether

in the Union list of additives which may be used in the manufacture of plastic materials and articles intended tocome into contact with foodstuffs under Directive 2002/72/EC (notified under document C(2010) 1613)



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5.4.4. Triclosan in animal husbandry

Triclosan is notified in the framework of the European regulations on biocides (Directive98/8/EC) for use in veterinary hygiene biocidal products.

5.4.5. Triclosan as feed preservative

According to Regulation (EC) 1831/2003 on additives for use in animal nutrition the use oftriclosan as preservative in feed is not authorised. The substance is not listed in thecorresponding Community Register of Feed Additives (2004/C 50/01).

5.5. Triclosan in the environment

The widespread use of triclosan results in the discharge of this compound to wastewater.Incomplete removal of triclosan from wastewater treatment plants (WWTPs) as well asspreading the triclosan laden biosolids into soils, leads to triclosan being distributed in soilsand surface waters.

Triclosan has been widely detected (see Table 4) in influents, effluents and biosolids ofWWTPs, in lakes, rivers and sea water in various countries in Europe (Paxeus 1996,Lindström et al . 2002, Adolfsson-Erici et al . 2002, Kanda et al . 2003, Bester 2003,Sabaliunas et al . 2003, Samsø-Petersen et al . 2003, Xie et al . 2008, Singer et al . 2002,Tixier et al .2002, van Stee et al . 1999, Kantiani et al . 2008, Dye et al . 2007), in the USA(McAvoy et al . 2009, Coogan et al . 2007, Coogan et al . 2008, US EPA 2009, Cha andCupples 2009, Fair et al . 2009, Halden and Paull 2005, Chalew and Halden 2009, Kumar etal . 2010), in Canada (Hua et al . 2005), in Australia (Ying and Kookana 2007, Fernandes etal . 2008), in Japan (Okumura and Nishikawa 1996) and in Hong Kong (Chau et al . 2008).

5.5.1. Fate of triclosan in the environmentBacteria are able to survive triclosan exposure by activating specific or general geneticcascades (see 6.2.4). The environmental concentrations of triclosan may affect bacterialactivities. Consequently it is important to evaluate the fate of triclosan in the environmentsuch as in WWTPs, rivers, effluents, etc.

Triclosan is transported through the domestic waste stream to WWTPs. Municipalwastewater treatment helps to achieve average removal efficiencies in the range of 58-99%, depending on the technical capabilities of sewage treatment systems (McAvoy et al .2002, Kanda et al . 2003, Bester 2003, Singer et al . 2002, Federle et al . 2002, Lishman etal. 2006, Lindström et al . 2002, Lopez-Avila and Hites 1980, Thomson et al . 2005, Ternes etal. 2004). However, mass balance studies have demonstrated that triclosan also exhibits

significant persistence, partitioning and sequestration in biosolids (by-product of wastewatertreatment). Approximately 50 ± 19% of the incoming mass of triclosan was observed topersist and become sequestered in biosolids produced by a conventional WWTPs employingactivated sludge treatment in conjunction with anaerobic biosolid digestion (Heidler andHalden 2007). Thus, important pathways of biocide release into the environment includeWWTP effluent discharge into surface waters and the land application of biosolids. Effluentfrom WWTPs contains a complex mixture of anthropogenic and natural compounds. Soilsamples from ten agricultural sites in Michigan previously amended with biosolids, collectedover two years, revealed triclosan concentration 0.16-1.02 µg/kg (Cha and Cupples 2009).90 to 7060 µg/kg triclosan was found in biosolids from 3 Michigan wastewater treatmentplants.

Triclosan, along with many other compounds, may have multiplicative or synergistic effects

on micro-organisms including bacteria.Environmental concentrations of triclosan reported in the published literature are describedin Table 4



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Table 4: Environmental concentrations of triclosan (data from worldwide sources)

Environmentalmatrix

Triclosanconcentration

Reference

Surface water

Lake/river/streamswith known inputof raw wastewater

1.4 ng/L-40000 ng/L Kolpin et al . 2002, Lindström et al . 2002,Lopez-Avila and Hites 1980, Singer et al .2002, Remberger et al . 2002, Kolpin et al .2004, Bendz et al . 2005, Glassmeyer et al .2005, Zhang et al . 2007, Halden and Paull2005, Chau et al . 2008, Coogan et al . 2007,Coogan and La Point 2008

20-86161 ng/L Lindström et al . 2002, Samsø-Petersen et al .

2003, Singer et al . 2002, Remberger et al .2006, McAvoy et al . 2002, 2009, Halden andPaull 2005, Lishman et al . 2006, Waltman etal . 2006, Heidler and Halden 2007, Kantiani etal . 2008, Fair et al . 2009, Kumar et al. 2009

Wastewater

Influent

Effluent23-5370 ng/L Lindström et al . 2002, Samsø-Petersen et al .

2003, Bester 2003; Kanda et al. 2003;Sabaliunas et al . 2003, Bendz et al . 2005,Halden and Paull 2005, Thompson et al . 2005,Ying and Kookana (2007), Fair et al . 2009,Kumar et al . 2009

Sea water <0.001-100 ng/L Xie et al . 2008, Okumura and Nishikawa 1996,

Fair et al . 2009

<100-53000 µg/kgd.w.

Fjeld et al . 2004, Remberger et al . 2002,Singer et al. 2002; Morales et al. 2005; Milleret al. 2008

Sediment

Lake/River/othersurface water

Marine 0.02-35 µg/kg d.w. Okumura and Nishikawa 1996, Fjeld et al . 2004

Biosolid fromWWTP 20-133000 µg/kgd.w. Svensson. 2002; Remberger et al . 2002,2006; Bester 2003; Morales et al. 2005;Kinney et al. 2006; Chu and Metcalfe 2007, USEPA 2009, Cha and Cupples 2009, Ying andKookana 2007

Activated/digestedsludge

580-15600 µg/kgd.w.

McAvoy et al . 2002, 2009, Singer et al . 2002,Chu and Metcalfe 2007, Kumar et al . 2010

Pore water 0.201-328.8µg/L(calculated)

Chalew and Halden 2009

d.w.: dry weight



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Photodegradation of Triclosan

Despite its high chemical stability, being extremely resistant to high and low pH, triclosan isreadily degraded in the environment via photodegradation. Eight photoproducts weretentatively identified, including chlorinated phenols, chlorohydroxydiphenyl ethers, 2,7- and

2,8-dichlorodibenzo-p-dioxin, and a possible dichlorodibenzodioxin isomer ordichlorohydroxydibenzofuran (Tixier et al . 2002; Sanchez-Prado et al . 2006a, 2006b,Canosa et al. 2005; Lores et al. 2005; Aranami and Readman 2007, Prada et al . 2004,Latch et al . 2005, Ingerslev et al. 2003). Some of these products show enhanced toxicitycompared to triclosan but have been shown to be degraded in the environment by bacteriasuch as Pseudomonas, Burkholderia and Sphingomonas (Field et al . 2008a and 2008b). Theend products are CO2 and chlorine with chlorocatechols as intermediates. Recently, Son etal . (2009) demonstrated that TiO2-photocatalytic degradation of triclosan is mainly achievedby radicals, and these radicals can further degrade dioxin-type intermediates once they areproduced in photocatalysis. The presence of hydrogen peroxide enhanced the oxidation (Yuet al . 2006).

Triclosan is hydrolytically stable under abiotic and buffered conditions over the pH 4-9 range

based on data from a preliminary test at 50°C. Photolytically, triclosan degrades rapidlyunder continuous irradiation from artificial light at 25°C in a pH 7 aqueous solution, with acalculated aqueous photolytic half-life of 41 minutes. One major transformation product wasidentified, 2,4-dichlorophenol, which was a maximum of 93.8-96.6% of the applied triclosan240 minutes after treatment.

Hydrolysis is not expected to be an important environmental fate process due to thestability of triclosan in the presence of strong acids and bases. However, triclosan issusceptible to degradation via aqueous photolysis, with a half-life of <1 hour under abioticconditions, and up to 10 days in lake water. An atmospheric half-life of 8 hours has alsobeen estimated based on the reaction of triclosan with photochemically produced hydroxylradicals. Additionally, triclosan may be susceptible to biodegradation based on the presenceof methyl-triclosan following wastewater treatment.

Degradation in chlorinated water

Triclosan addition to chlorine spiked ultra-pure water or to chlorinated tap water led to theformation of two tetra- and one penta-chlorinated hydroxylated diphenyl ether, as well as2,4-dichlorophenol. Chlorination of the phenolic ring and cleavage of the ether bond wereidentified as the main triclosan degradation pathways (Canosa et al . 2005). Free chlorinemediated oxidation of triclosan leads to the formation of chloroform and other chlorinatedorganics (Rule et al . 2005, Fiss et al . 2007).

Ozone treatmentTreatment with ozone during municipal sewage treatment was efficient at removal oftriclosan (Suarez 2007; Wert et al . 2009; Dodd et al . 2009). The degradation products werehowever not identified.

Biodegradation

Aerobic bacterial hydrolysis plays an important role in triclosan degradation. A consortium ofbacteria able to partially degrade triclosan was isolated and one consortium member wasshown to be a Sphingomonas-like micro-organism (Hay et al . 2001). In a different study,two strains of Pseudomonas putida TriRY and Alcaligenes xylosoxidans subsp. denitrificans TR1 were shown to utilise triclosan as sole carbon source (Meade et al . 2001). Zhao (2006)also isolated one strain of triclosan-degrading bacteria (Sphingomonas or Sphingopyxis)from activated sludge. Zhao also found that Nitrosomonas europaea, an importantnitrification bacterium in wastewater treatment plants has the ability to degrade triclosan



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through co-metabolism. Triclosan and its chlorinated degradation products can also bedegraded by bacteria (Pseudomonas, Sphingomonas, Burkholderia) under aerobicconditions.

Very little is known of the biochemistry of the biodegradation of triclosan and nothing is

documented in the Minnesota biodegradation database (http://umbbd.msi.umn.edu/).There is a data gap on the degradation pathway of triclosan and its intermediary products.

Under anaerobic conditions and in the dark, triclosan is quite stable. Due to its low watersolubility, triclosan is readily adsorbed to particles and tends to accumulate in sediments.Digested sludge concentrations of triclosan ranged from 0.5 to 15.6 µg/g (dry weight),where the lowest value was from an aerobic digestion process and the highest value wasfrom an anaerobic digestion process. These results suggest that triclosan is readilybiodegradable under aerobic conditions, but not under anaerobic conditions (McAvoy et al .2009).

The limited data available indicate that effect levels of triclosan on activated sewage sludgemicro-organisms vary depending on the level of acclimation. A concentration of 2 mg/L

inhibited activated sludge micro-organisms that had not been acclimated to triclosan;however, the same concentration had no effect on acclimated organisms. Laboratory-derived IC50 values range from 20-239 mg triclosan/L based on carbon dioxide (CO2)evolution and glucose utilisation.

Triclosan (≥2 mg/L) had a slight effect on chemical oxygen demand removal underlaboratory conditions, but had a major inhibitory effect on the nitrification process.Anaerobic sludge digestion was significantly inhibited at a concentration of 10 mg/L. A NOECfor sewage microbes was not available (NICNAS 2009).

5.5.2. Effect of triclosan on micro-flora and toxicity of metabolites

Inhibitory effects on micro-organisms were shown to begin at concentrations ranging from

25 to 80,000 µg ⁄ L for triclosan (Federle et al . 2002, Samsø-Petersen et al . 2003, Sivaramanet al . 2004, Neumegen et al . 2005, Stasinakis et al . 2007, Farre et al . 2008, Stickler andJones 2008). It should be noted that the upper range minimum inhibitory concentrations(MICs) reported are well in excess of published solubility limit for triclosan. MIC thresholdvalues for micro-organisms are exceeded by environmental levels of triclosan in several

sediments, biosolids, and activated sludge. Lawrence et al . (2009) observed a change in thestructure and composition of a river biofilm microcosm following exposure to triclosan (10µg/L) over a 8-week period.

Waller and Kookana (2009) studied the effect of triclosan on selected microbiologicalactivity and biochemical parameters in Australian soil. Substrate-induced respiration andnitrification, plus activities of four enzymes relevant for carbon turnover (acid and alkali

phosphatase, 3-glucosidase, and chitinase) were measured. The effect of triclosan onenzymatic activity was minimal even at a high concentration (100 mg/kg). Likewiserespiration was not affected. However, the study demonstrated that triclosan atconcentrations below 10 mg/kg can disturb the nitrogen cycle in some soils.

McBain et al . (2003) showed that long-term exposure of domestic-drain biofilms tosublethal levels of triclosan (2-4 g/L, four times daily) did not affect bacterial viability orsignificantly alter antimicrobial susceptibility. This lack of effect may reflect the biofilmphenotype present in the microcosm, the presence of intrinsically tolerant bacteria anddegradation of triclosan by the drain biofilm consortium. However, microbial diversity afterexposure to triclosan was profoundly affected.

Studies reporting on the effect of repeated exposure of triclosan against complex oral

microcosms failed generally to show an increase in resistance determined either by anincrease in MIC or in Minimal Bactericidal Concentration (MBC) (Sullivan et al . 2003; McBainet al . 2004). In addition, McBain et al . (2004) did not observe any cross-resistance to otherbi id ibi i ( li d i di l ) i b f b i l

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species such as Streptococcus sanguis, Sterptococcus oralis and Prevotella nigrescens witha decreased susceptibility to triclosan resulting from exposure to the bisphenol. However,these results contrasted with those obtained with E. coli , for which repeated exposure toincreasing concentrations of triclosan led to a 400-fold increase in resistance (MBC from 0.2to 39.1 mg/L) (McBain et al . 2004). Moreover, bacteria inside biofilms resist better to

biocidal agents. For example, reduced susceptibility to triclosan was observed in Salmonella(Tabak et al . 2007) and Proteus/Providencia (Stickler and Jones 2008, Williams and Stickler2008).

5.6. Triclosan in the human body

Triclosan enters the human body orally through toothpaste, mouthwashes and dentaltreatments. In humans, triclosan is rapidly and completely absorbed from thegastrointestinal tract, while a lower rate of absorption occurs dermally. It has been found inhuman blood, plasma and milk (Allmyr et al . 2006, 2008, Adolfsson-Erici et al . 2002) inSweden and Australia. In the USA it was found in human urine (Calafat et al . 2008). A

volunteer study in Sweden (Sandborgh-Englund et al . 2006) showed that the accumulatedurinary excretion varied between the subjects, with 24 to 83% of the oral dose beingexcreted during the first 4 days after exposure.

6. MECHANISMS OF RESISTANCE TO TRICLOSAN

6.1. General considerations on biocide resistance in bacteria

Unlike antibiotic resistance, the issues relating to biocide resistance in the healthcareenvironment are considered to have a very low profile and priority (Cookson 2005). Despitethe widespread use of disinfectants and antiseptics in healthcare settings, acquiredresistance to biocides in bacteria isolated from clinical specimens or the environment is notroutinely characterised. Emerging bacterial resistance to biocides has been well described invitro, but evidence in practice is still lacking (Russell 2002b, Cookson 2005, Maillard andDenyer 2009).

It is widely accepted that biocides have multiple target sites against bacteria (Denyer andMaillard 2002, Lambert 2002, Maillard 2002, Maillard 2007, Poole 2004, Stickler 2004,Gilbert and Moore 2005, Maillard 2005b)with their efficacy depending on a range of intrinsicand extrinsic factors, (Reuter 1984, 1989, 1994, EFSA 2008, SCENIHR 2009). Thus, theemergence of general bacterial resistance is likely to arise from a mechanism/processcausing the decrease of the intracellular concentration of a biocide, under the threshold that

is harmful to the bacterium (Tattawasart et al. 2000a, Tattawasart et al. 2000b; Braoudakiand Hilton 2005; Maillard 2005a, Maillard and Denyer 2009). Several mechanisms based onthis principle (mode of action) have been described including change in cell envelope,change in permeability, efflux and degradation (Table 5). Bacteria in biofilms are also lesssusceptible to biocides because of a number of factors. It is likely that some of thesemechanisms operate synergistically although very few studies investigating multiplebacterial mechanisms of resistance following exposure to a biocide have been performed.

Bacterial resistance to biocides is not a new phenomenon and it has been reported since the1950’s (Adair et al. 1971; Russell 2002b; Chapman 2003). To date, bacterial resistance hasbeen described for all the biocides that have been investigated. Resistance often occursfollowing an improper usage of the formulated biocide, leading notably to a decrease inactive concentration (Sanford 1970, Prince and Ayliffe 1972, Russell 2002b).



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Table 5: Mechanisms of bacterial resistance to biocides at the cell level

Mechanisms References

Decrease in concentration (thatreaches the target sites)

Spores (layers: cortex, sporeenvelope)

Russell 1990; Russell et al. 1997;

Denyer and Maillard 2002; Lambert2002; Cloete 2003, Hawkey 2004;Champlin et al. 2005;

Gram-negative (outer membrane)- Lipopolysaccharides

- Proteins (porins)

- Fatty acid- Phospholipids

Munton and Russell 1970; Ayres etal . 1998; McDonnell and Russell1999; Tattawasart et al. 2000a, b;Denyer and Maillard 2002; Fraud et

al. 2003; Stickler 2004; Braoudakiand Hilton 2005Gandhi et al. 1993; Brözel andCloete 1994; Winder et al. 2000

Jones et al. 1989; Méchin et al. 1999; Guérin-Méchin et al. 1999,2000Boeris et al. 2007

Change incell

permeability

Mycobacteria mycoylarabinagalactan McNeil and Brennan 1991; Broadleyet al . 1995; Russell, 1996; Russellet al . 1997; Manzoor et al. 1999;Walsh et al. 2001; Lambert, 2002

Change insurfaceproperties

Decrease binding and interactionbetween biocide and cell surfacesSurface charge

Bruinsma et al. 2006

Efflux

mechanisms

Decrease intracellular concentration

of a biocide- Small multidrug resistance (SMR)family (now part of thedrug/metabolite transporter (DMT)superfamily)

- Major facilitator superfamily (MFS)- ATP-binding cassette (ABC) family- Resistance-nodulation-division

(RND) family- Multidrug and toxic compound

extrusion (MATE) family

Nikaido, 1996; Paulsen et al. 1996;

Schweizer 1998, 2001; Brown et al. 1999; Putman et al. 2000; Borges-Walmsley and Walmsley, 2001;Poole, 2001, 2002a, b; Levy 2002;Chuanchuen et al . 2003; McKeeganet al . 2003; Piddock 2006

Enzymaticmodification

Decrease intracellular and exocellularconcentration of a biocide

Demple 1996; Kummerle et al .1996; Valkova et al . 2001; Cloete2003;

Targetmutation

FabI mutation in Mycobacteriumsmegmatis

McMurry et al . 1999;

By-passmetabolicblockage

Increase in pyruvate synthesis andfatty acid production via an alteredmetabolic pathway (expression of

‘triclosan resistance network’)

Webber et al . 2008b

It is worth noting that some mechanisms (e.g. efflux, target protection, degradation) can behorizontally transferred to other bacteria (Poole 2002a, Quinn et al . 2006, Roberts and

Mullany 2009, Yazdankhah et al . 2006; Hawkey and Jones 2009, Juhas et al . 2009). Inaddition, Pearce et al . (1999) showed that some biocides, at a sub-lethal concentration,may increase or decrease the frequency of gene transfer by conjugation and transduction



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6.2. General considerations on the study of triclosan

Triclosan is described as a broad spectrum biocide. However, some bacteria are intrinsically

resistant to triclosan, notably P. aeruginosa (Lear et al . 2002) and triclosan is not activeagainst bacterial endospores. This is likely due to the structure of the Gram-negativebacteria and particularly the outer membrane, preventing triclosan to penetrate through thebacterium to reach its target sites.

Bacterial resistance mechanisms to triclosan have been widely studied. However, moststudies have considered resistance as an increase in MIC and not necessarily as an increasein MBC. Using MICs to measure bacterial resistance is arguable, since much higherconcentrations of biocides have usually been used in practice and, therefore, failing toachieve lethality because of elevated MICs is unlikely. Some studies have shown thatbacterial strains showing a significant increase in MICs to some biocides, such as cationics,were nevertheless susceptible to higher (in use) concentrations of the same biocide

(Thomas et al. 2005) or triclosan (Lear et al. 2006). MRSA showing a 40-fold increase inMIC to triclosan remained susceptible to 1 mg/L (Suller and Russell 1999). Concentration iscentral to the definition of resistance in practice (Maillard and Denyer 2009). Hence,bacterial resistance based on the determination of MIC does not reflect accurately the trueresistance profile of biocides, including triclosan.

Concentration is one the most important factors that will affect the activity and efficacy of abiocide (Russell and McDonnell 2000, Maillard 2005a, b 2007). Biocides with a highconcentration exponent (Russell and McDonnell 2000) such as triclosan are particularlyaffected by dilution since a small decrease in concentration will profoundly affect efficacy.Hence, it might not be surprising that products with a low concentration of a phenolicbiocide or other biocides with a high concentration exponent (e.g. alcohols) are lesseffective and might be prone to bacterial contamination and growth.

Most laboratory studies have been performed with triclosan dissolved in a solvent such asDMSO, and in some cases alcohol, and did not investigate commercially availableformulations. Differences between laboratory (in vitro) investigations and situations inpractice have not been addressed to date (Maillard and Denyer 2009). Hence, emergingbacterial resistance to triclosan investigated in vitro conditions might not necessarily reflectsuch development of resistance in situ. Components of the formulations might have apotentiation effect (or not) on the activity of triclosan, and their role on emerging bacterialresistance to triclosan has not been studied.

6.3.

Mechanisms of bacterial resistance to triclosanBacterial resistance against triclosan involves both intrinsic and acquired mechanisms(Yazdankhah et al . 2006), and include: mechanical barrier (altering intracellularconcentration), change in target site (mutation of the target site) (Heath et al . 1998),efflux, and by-pass of metabolic pathway (Webber et al . 2008b). These mechanisms havebeen also described to confer antibiotic resistance (Davin-Regli et al. 2008).

Change in enoyl acyl carrier reductase

At sub-lethal concentrations, triclosan has been shown to affect specific bacterial targets.Triclosan interacts specifically with an enoyl-acyl reductase carrier protein (ENR) at a lowconcentration (Heath et al . 1999; Levy et al . 1999, Roujeinikova et al . 1999, Stewart et al .1999). Triclosan was found to inhibit fatty acid synthesis by targeting FabI in E. coli (Heathet al . 1998) and S. aureus (Heath et al . 2000), and InhA in M. smegmatis (McMurry et al .1999) and M tuberculosis (Parikh et al 2000)



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Triclosan resistant mutations in fabI decrease the interaction of triclosan with the ENR-NAD+ binding. Mutation in fabI in E. coli was shown to confer a 60-fold decreased susceptibility totriclosan (Heath et al . 1998). Mutation in fabI has led to an increase in triclosan MIC in anumber of bacterial genera (McMurry et al . 1998a, Parikh et al . 2000, Health et al . 2000,Slater-Radosti et al . 2001, Massengo-Tiassé and Cronan 2008, Webber et al. 2008b). In

Acinetobacter baumannii high-level triclosan resistance could be explained by a Gly95Sermutation of FabI, whilst wild-type fabI was observed to be overexpressed in low-levelresistant isolates (Chen et al . 2009). Likewise in Ps. aeruginosa, high-level resistance totriclosan has been shown to be associated with FabV (Zhu et al. 2010).

McMurry et al . (1998b) postulated that mutations at mar and sox in E. coli only conferred a2-fold increase in resistance presumably by a modest overexpression of AcrAB. Thisexpression is unlikely to decrease the efficacy of triclosan. However such a mutation,together with mutations at other loci such as fabI (increasing resistance to 90-140-fold)could be more significant

Efflux of antimicrobialsTriclosan is a substrate of AcrAB efflux pump in E. coli , of MexAB-OprM and MexCD-OprJ,MexEF-OprN, MexJK-OprH multidrug efflux pumps in P. aeruginosa, of AcrB in S. enterica serovar Typhimurium, and CmeB in Campylobacter spp. (Piddock 1996; McMurry et al .1998; Chuanchuen et al . 2001, 2002, 2003; Schweizer 1998). These efflux pumps aresimilar to other efflux pumps in other Gram-negative pathogens (Piddock 2006) and assuch, it is likely that triclosan is a substrate of such pumps in other Gram-negative bacteria.

In S. enterica serovar Typhimurium, active efflux via AcrAB-TolC conferred decreasedsusceptibility to triclosan. The triclosan resistant mutants (MIC ≥32 mg/L) did not lose anyfitness when compared to wild-type strains (Webber et al . 2008a). The pump efflux systemof P. aeruginosa has been shown to confer a high level of intrinsic triclosan resistance (Mimaet al . 2007). In addition, mutants of E. coli , and S. enterica which overexpress the AcrAB–TolC efflux system, have decreased susceptibility to various agents, including triclosan,demonstrating that triclosan is a substrate for efflux pumps (Webber et al . 2008a).

As previously reported for antibiotics, the presence of active efflux pumps is required for theacquisition of target mutations, which in turn increase the level of resistance (Webber et al. 2008b). In Acinetobacter baumannii , although active efflux did not appear to be a majorreason for triclosan resistance, the acquisition of resistance appeared to be dependent on abackground of intrinsic triclosan efflux (Chen et al . 2009).

By-pass of metabolic blockage

The proteomic analysis of S. enterica serovar Typhimurium triclosan-resistant mutants

showed a set of proteins with commonly altered expression in all resistant strains. This “triclosan resistance network” included 9 proteins involved in production of pyruvate or fattyacid and represents a mechanism to increase fatty acid synthesis by an alternative pathway(Webber et al . 2008b). In addition to the expression of this “network”, these mutantsshowed specific patterns of protein expression leading to the conclusion that triclosanresistance was multifactorial and potentially involved a number of mechanisms actingsynergistically to attain high-level resistance (≥32 mg/L) (Webber et al . 2008b). In S.

aureus, a modification of the membrane lipid composition associated with the alteration ofthe expression of various genes involved in the fatty acid metabolism were observed intriclosan resistant strains (Tkachenko et al . 2007).

Seaman et al . (2007) studied the appearance of small colony variants in MRSA followingexposure to triclosan in vitro. The small colony variants displayed reduced susceptibility(23-60 fold; 1.5-4 mg/L from 0.063 mg/L) to triclosan and resistance to penicillin andgentamicin. Bayston et al. (2009) noted that prolonged exposure (i.e. 72 h) to triclosan-



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impregnated silicone resulted in the induction of small colony variants and a 67-foldincrease in triclosan MIC.

Recent evidence highlighted that bacterial swarming motility might confer some resistanceto triclosan (5 mg/mL in B. subtilis and 0.1 mg/mL in E.coli ) when compared to no

swarming bacteria. The mechanism(s) by which swarming might confer some resistance isunknown, but is unlikely to be caused by efflux (Lai et al . 2009).

Involvement of multiple mechanisms

At bactericidal concentrations, triclosan seems to act against multiple and various targets,causing disruption of the bacterial control of cell wall permeability (Villalain et al . 2001;Guillén et al . 2004). One study in particular, investigated the role of both the permeabilitybarrier and efflux in increase resistance to triclosan in E. coli . The MIC of triclosan-resistantE. coli mutants (MIC >1000 mg/L) was reduced to 10-25 mg/L when treated with bothethylene diamine tetra-acetic acid (EDTA; a chelating agent enhancing outer membranepermeability) and carbonyl cyanide m-chlorophenylhydrazone (CCCP; a proton motive force

uncoupler), as compared to a MIC of 0.1 mg/L in sensitive E. coli strain, indicating thatpotentially both permeability and efflux worked together to provide the high level resistanceto triclosan. However, neither CCCP nor EDTA reduced the susceptibility of P. aeruginosa totriclosan (Gomez Escalada 2003). In Acinetobacter baumannii , triclosan-resistant isolateswere characterized by antibiotic susceptibility, clonal relatedness, fabI mutation, fabI expression, and efflux pump expression (Chen et al . 2009). Yu et al. (2010) described amultiple mechanism response in E. coli following exposure to triclosan. The involvement of anumber of mechanisms was shown to confer triclosan resistance up to 80 mg/L.

Bacterial biofilms

Generally, bacteria are attached to surfaces and associated in a community (termed biofilm)

and are rarely present as single cells (planktonic). Bacterial biofilms have been shown to behighly resistant to antimicrobials compared to planktonic cultures. A biofilm-associatedphenotype has been described (Brown and Gilbert 1993, Ashby et al . 1994, Das et al . 1998;Gilbert et al . 2003). The mechanisms of resistance involved in a bacterial biofilm includedecreased metabolism, quiescence, reduced penetration due to the extracellular polymericmatrix (Pan et al . 2006), enzymatic inactivation of biocides (Sondossi et al . 1985)Giwercman et al . 1991, Huang et al . 1995), and the induction of multi-drug resistantoperons and efflux pumps (Maira-Litran et al . 2000). Bacterial biofilm resistance to triclosanhas been poorly studied.

One study reported that the tolerance to triclosan of Salmonella in biofilm was attributed tolow diffusion through the extracellular matrix, while changes of gene expression might

provide further resistance both to triclosan and to other antimicrobials (Tabak et al . 2007).McBain et al . (2003) investigated the fate of a complex bacterial biofilm exposed to sub-lethal concentrations of triclosan (2–4 g/L) over a 3 month period. The authors identified achange in the composition of the biofilm and an increase in resistance of the complexpopulation as measured by MIC. Interestingly, the composition of the biofilm changed, witha decrease of species diversity. The triclosan tolerant species such as Pseudomonads andStenotrophomonads were still present, but other triclosan tolerant species ( Achromobacter

xylosoxidans) demonstrated a clonal expansion. Most importantly, the authors noted thatthe antibiotic susceptibility profile was not affected.

A study investigating the effect of triclosan in the development of bacterial biofilms onurinary catheters highlighted the selectivity of triclosan. While triclosan inhibited P.mirabilis, it had little effect on other common bacterial pathogens (Jones et al. 2006). In

addition, the control of P. mirabilis by triclosan resulted in emerging triclosan-resistantstrains in vitro. While most of these strains were still susceptible to the triclosanconcentration used in the urinary catheter, one strain (MIC = 40 mg/L) was not (Stickler



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three biocidal products used in healthcare (one containing benzalkonium chloride 10 g/L,one containing chlorhexidine gluconate 40 g/L and one containing triclosan 10 g/L), wereineffective in eliminating hospital-acquired MRSA or P. aeruginosa biofilms, highlightingdifferences in susceptibility between planktonic and biofilm bacteria.

It is however interesting to note that Tabak et al. (2009) observed a synergistic action ofsequential treatment of triclosan (500 mg/L) followed by ciprofloxacin (500 mg/L) againstbiofilm of S. enterica serovar Typhimurium. There is little information in the literature aboutthe potentiation of activity between a biocide and an antibiotic and such a study isimportant and describes an interesting application/effect of triclosan.

6.4. Mutation rates and transfer of resistance

The development of bacterial resistance through acquired mechanisms such as mutationand the acquisition of resistant determinants are of concern since a bacterium that was

previously susceptible can become insusceptible to a compound or a group of compounds(Russell 2002a). In S. enterica serovar Typhimurium, mutation frequency followingexposure to triclosan was low (5 x 10-9), lower than mutation frequency observed followingantibiotic exposure (Birošová and Mikulášová 2009).

Cookson et al . (1991) isolated MRSA strains exhibiting triclosan resistance (2-4 mg/L) frompatients using mupirocin and triclosan baths. Although in this study the resistance wasshown to be transferable in association with the plasmid encoding for mupirocin resistance,this could not be confirmed subsequently by other studies. The transfer of a plasmidencoding for mupirocin resistance to a triclosan sensitive S. aureus strain failed to increaseresistance to triclosan (Suller and Russell 2000). Other studies investigating clinical S.aureus isolates resistant to mupirocin also failed to observe this association (Bamber andNeal 1999). Although various genetic mobile elements have been described to be involved

in the dissemination of cross-resistance towards biocides-antibiotics (Roberts and Mullany2009, Schlüter et al . 2007) no specific genetic mobile element has been associated withtriclosan resistance.

6.5. Induction of resistance

There are two types of induction. The first corresponds to the trigger of genes governing thegenetic cascade (global regulation) which promotes the expression of efflux pumps and/ordown regulates membrane permeability (porin synthesis). The second corresponds to thedirect activation of the promoter region (local regulation) for example controlling efflux

genes (Davin-Regli et al . 2008).The induction of bacterial resistance mechanisms following exposure to a low concentrationof a biocide has been reported in a number of studies for a number of biocides (SCENIHR2009). In some occasions, a specific mechanism has not been established and a phenotypicchange leading to the emergence of resistance to several unrelated compounds in vitro hasbeen reported following exposure to a low concentration of a biocide (Moken et al . 1997).

It is possible that triclosan induces a stress response followed by, or in addition to, theexpression of mechanisms that reduce the deleterious effect of the biocide (McMurry et al .1998b; Gilbert et al . 2002). A decrease in growth rates in E. coli and P. aeruginosa hasbeen described following exposure to sub-lethal concentrations of triclosan, which indicatesthe generation of a stress to the organism (Gomez Escalada et al . 2005).

Triclosan induces bacterial resistance through the over-expression of efflux pumps viaactivation of mar and ram (Randall et al. 2007; Webber et al . 2008a; Bailey et al. 2009),over-expression and mutagenesis of fab1, expression of regulatory genes involved in the



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permeability) and fatty acid metabolism in a number of bacterial genera (Jang et al . 2008,Webber et al . 2008b, Bailey et al . 2009). These genes are involved in resistance totriclosan, but also in possible cross-resistance and multi-resistance to different antibioticand biocide classes. In Stenotrophomonas maltophilia, the overexpression of an efflux pump(SmeDEF), involved in antibiotic resistance, was demonstrated in several triclosan-selected

mutants (Sánchez et al . 2005). In E. coli , overexpression of acrAB or marA or soxS (positiveregulator of acr AB) decreased susceptibility to triclosan 2-fold. Deletion of the acr AB locusincreased susceptibility to triclosan approximately 10-fold. It was observed that clinicalisolates overexpressing acrAB showed enhanced resistance to triclosan. A clinical strainoverexpressing marA had a triclosan MIC of 0.27mg/L as compared to susceptible strainwith an MIC of 0.090 mg/L. In S enterica serovar Typhimurium overexpressing AcrAB andC. jejuni overexpressing CmeB, triclosan MIC increased to 32 mg/L (Pumbwe et al . 2005;Buckley et al . 2006). Moken et al . (1997) described the induction of the MDR phenotype inE. coli and its relevance to cross-resistance between pine oil, triclosan and multipleantibiotics. Jang et al . (2008) reported that, in S. aureus, exposure to triclosan (0.015mg/L) resulted in down-regulation of the clpB chaperone-related genes, which might triggerthe expression of resistant determinants. A recent study demonstrated that triclosan

activates the expression of several groups of genes in E. coli and S. enterica (Bailey et al 2009). Transcriptome analyses (including microarray and RT-PCR experimental approaches)of bacteria exposed to triclosan (0.12 mg/L for 30 minutes) indicated an induction of theexpression of various genes involved in drug efflux (e.g. acrB), in the genetic activation ofresistance genes (e.g. marA), in the control of oxidative and drug response (e.g. soxS), andin the control of membrane permeability (e.g. ompR). Despite some differences in theresponse level observed between the two bacterial species, triclosan was shown to induce arapid and adaptative response including the activation of several regulatory and structuralgenes involved in antibiotic resistance (Bailey et al. 2009).

McBain et al . (2004), however, failed to demonstrate a biologically significant induction ofdrug resistance in a number of bacterial species exposed to sub-lethal concentrations oftriclosan, suggesting that triclosan-induced drug resistance is not generally readily induciblenor is it transferred across bacterial species.

6.6. Bacterial cross-resistance to triclosan and antibiotics

6.6.1. General considerations

The possibility that the mechanisms involved in triclosan resistance may contribute toreduced susceptibility to clinically important and structurally unrelated antimicrobials is ofmajor concern. It is important to note that antibacterial actions from antibiotics and biocides

show some similarities in their mechanisms of action, behaviour and clinical aspects (Poole2007).

Among the similarities, we can mention (i) the penetration/uptake through bacterialenvelope by diffusion, (ii) the effect on the membrane integrity and morphology, (iii) theeffect on diverse key steps of bacterial metabolism (replication, transcription, translation,transport, various enzymes). Faced with this chemical aggression and stress, bacteriamobilise similar defence mechanisms conferring resistance against structurally non-relatedmolecules (Walsh and Fanning 2008).

6.6.2. Triclosan and cross-resistanceA number of (but not all) laboratory studies have demonstrated an association betweentriclosan resistance and resistance to other antimicrobials However this link has not been



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confirmed in the limited number of in situ studies that have been performed to date. Anumber of bacterial mechanisms potentially conferring cross-resistance has been identifiedin laboratory investigations (see Table 6).

Table 6 Bacterial mechanisms inducing potential cross-resistance

Mechanism Nature Level ofsusceptibility toother biocides1

Cross-resistance

Change in bacterial envelope intrinsic (acquired) no yes

(over)Expression of effluxpumps

intrinsic/acquired reduced yes

Enzymatic modification acquired/intrinsic reduced no2

Mutation (target site) acquired reduced no3

Phenotypic change Following exposure reduced yes1 to other biocides - level of susceptibility defined according to the concentration of biocides2 in the case of acquired resistance, co-resistance has been described3 triclosan cross-resistance with specific antibiotics (e.g. isoniazid) acting against enoyl acyl carrier proteins (e.gFabI) has been described.

Studies on S. enterica and Stenotrophomonas maltophilia described the effect of triclosanon emerging bacterial cross-resistance. In S. enterica, Karatzas et al . (2007) reported thata triclosan-resistant strain overexpressing an efflux pump was less susceptible to antibioticsthan the wild type original strain. Another study described the survival of S. enterica serovar Typhimurium following exposure to various disinfectants at a low concentration onthe resulting changes in antibiotic profile (Randall et al . 2007). The authors concluded that

growth of Salmonella with sub-inhibitory concentrations of biocides favours the emergenceof strains resistant to different classes of antibiotics. In Stenotrophomonas, Sanchez et al .(2005) analysed the effect of triclosan on the selection of mutants overexpressing the effluxpump SmeDEF involved in both intrinsic and acquired resistance to antibiotics. The authorsdemonstrated that triclosan was able to select 5 mutants overexpressing this pump, out of atotal of 12 mutants. This overexpression conferred resistance to a number of antibioticssuch as tetracycline, chloramphenicol and ciprofloxacin.

Similar results have been reported with S. enterica and E. coli (Braoudaki and Hilton 2004).E. coli O157 strains, involved in the "hamburger disease", acquired high- levels of resistanceto triclosan after only two sublethal exposures and when adapted, repeatedly demonstrateddecreased susceptibilities to various antibiotics, including chloramphenicol, erythromycin,

imipenem, tetracycline, and trimethoprim, as well as to a number of biocides. Bailey et al .(2009) showed that triclosan triggered the expression of a number of genes (e.g. encodingfor efflux pumps, porins) directly involved in antibiotic resistance, and regulatory genesinvolved in the control of the antibiotic resistance gene cascade (activator of drug efflux,decrease of membrane permeability). Alteration in InhA in M. smegmatis following exposureto triclosan resulted in resistance to isoniazid (McMurry et al . 1999). Likewise, exposure ofM. tuberculosis to triclosan led to mutation in inhA causing cross-resistance to isoniazid.However, isoniazid-resistant mutants were still susceptible to triclosan (Parikh et al . 2000).

Pycke et al. (2010) observed that triclosan exposure of the environmental α-proteobacterium Rhodospirillum rubrum led to an increase in triclosan MIC. The extent ofthis increase as well as the generation of different antibiotic susceptibility profiles wastriclosan-concentration dependent, indicating the expression of distinct resistance

mechanisms.However, direct linkage between triclosan usage and bacterial resistance to other biocidesand antibiotics might not be universal Cottell et al (2009) investigated the antibiotic



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susceptibility of triclosan tolerant S. aureus, E. coli and Acinetobacter johnsonii and reportedthat these strains remain susceptible to antibiotics used in clinical settings. In addition,triclosan-tolerant E. coli were found to be significantly more susceptible to aminoglycosides(Cottell et al . 2009). Likewise, triclosan resistant mutants in S. aureus did not show analtered antibiotic susceptibility profile compared to their parent strains (Suller and Russell

2000). Lear et al . (2006) demonstrated that environmental isolates with an increased MICto triclosan remained susceptible to other biocides and antibiotics. Birošová and Mikulášová(2009) reported that continuous exposure of sub-inhibitory concentrations of triclosan didnot increase emerging antibiotic resistance in S. enterica serovar Typhimurium but helpedto maintain antibiotic-resistant bacteria in the population, notably those showing a mar phenotype. A short-term exposure to triclosan (30 min at 0.5 MIC, i.e. 0.098 mg/L) did notresult in the selection of antibiotic resistant mutants.

6.7. Triclosan resistance in bacteria in situ

Triclosan has been the most studied biocide with respect to its anti-bacterial activity.However, investigations concerned mainly laboratory experiments and only very few studiesare available to date on bacterial resistance to triclosan in situ. Furthermore, in most invitro studies, resistance to triclosan has been measured as an increase in MIC. Asmentioned in section 6.2 above, the measurement of resistance based on MIC only, mighthave little bearing on bacterial survival to concentrations found in situ.

Ledder et al . (2006) investigated acquired high-level triclosan resistance in a number ofdistinct environmental isolates and reported that a relatively small number of strainsshowed a decrease in triclosan susceptibility (E. coli , Klebsiella oxytoca, Aranicola

proteolyticus and S. maltophilia) while the susceptibility of the remaining 35 speciesremained unchanged. They concluded that repeated exposures to triclosan did notsystematically produce high-level triclosan resistance in all bacteria. Furthermore, amongthe strains with decreased susceptibility, there was no change in antibiotic susceptibility orsusceptibility to other biocides. Similarly, another study by the same group on repeatedexposure of dental bacteria to triclosan resulted in the same conclusions (McBain et al .2004).

Cole et al . (2003) collected 1238 isolates from the homes of users and non-users ofantibacterial product and were unable to demonstrate any cross-resistance to antibiotic andantibacterial agents in target bacteria. In addition, this study showed an increasedprevalence of potential pathogens in the homes of non-users of antibacterial products.However, in this study, the isolates were selected based on their antibiotic resistance andwere then tested for their insusceptibility to biocides. With our current state of knowledge, itis generally accepted that antibiotic resistance in clinical isolates is not necessarily

associated with resistance to biocides. Sullivan et al . (2003) studied the effect of triclosan intoothpaste on some bacterial species from the oral flora of 9 human volunteers over a 14-day period. Triclosan usage contributed to a decrease in lactobacilli although this decreasehad no clinical significance. Furthermore, the antibiotic susceptibility profile of the oralstreptococci investigated did not change following the use of triclosan containing toothpaste.Aiello et al . (2004) did not find any statistical significance between elevated triclosan MICsand antibiotic susceptibility in bacterial isolates taken from the hands of individuals usingantibacterial cleaning and hygiene products for a 1-year period. Earlier studies reported nochange in the ecology of the oral flora or resistance to triclosan following the use oftriclosan-containing toothpaste. Jones et al . (1987) reported no change in the predominantplaque flora in 13 volunteers following the use of triclosan (2 g/L) for seven months. Theauthors did not observe any increase in triclosan MIC in these bacteria. Similar conclusionswere reported by Walker et al . (1994) who reported no changes in the microbial flora in 144patients following the use of 3 g/L triclosan-containing toothpaste. A meta-analysis of 16clinical studies of the long-term effect (at least 6 month) of using triclosan toothpasteshowed reduction in dental plaques and gingivitis (Davies et al 2004)



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7. TRICLOSAN BIOAVAILABILITY AND FORMULATION EFFECTS

The concentration of triclosan that comes in contact with a micro-organism governs thesubsequent effect on that micro-organism (e.g. inhibitory, lethal, adaptation, selection).Hence the bioavailability of triclosan is paramount.

As described in Chapter 5, triclosan present in various environmental media is susceptible todegradation by oxidation by ozone, chlorine and sunlight, and to biodegradation by micro-organisms. The main route of exposure to soil is expected to be via the application ofsewage sludge to agricultural soil. The bioavailability will depend on the sorption, mobilityand degradation in soil under various physical conditions. Triclosan is released into surfacewaters via effluents from WWTP, and the bioavailability of the triclosan to micro-organismsin these media will depend upon sedimentation by binding with the particulate matter andstability of the compound during the exposure period.

The US EPA (2008) states on stability of triclosan in the environment that:

"Triclosan is hydrolytically stable under abiotic and buffered conditions over the pH 4-9range based on data from a preliminary test at 50°C.

Photolytically, triclosan degrades rapidly under continuous irradiation from artificial light at25°C in a pH 7 aqueous solution, with a calculated aqueous photolytic half-life of 41minutes.

Triclosan degrades rapidly in aerobic soils maintained in darkness at 20 ± 2°C, withcalculated half-lives of 2.9-3.8 days.

In aerobic water-sediment systems maintained in darkness at 20 ±2°C, triclosan degradedwith calculated nonlinear half-lives of 1.3-1.4 days in the water, 53.7-60.3 days in the

sediment, and 39.8-55.9 days in the total system.In soil, triclosan is expected to be immobile based on an estimated Koc of 9,200.

Triclosan is not expected to volatilize from soil (moist or dry) or water surfaces based on anestimated Henry’s Law constant of 1.5 x 10-7 atm-m3 /mole.

Triclosan partially exists in the dissociated form in the environment based on a pKa of 7.9,and anions do not generally adsorb more strongly to organic carbon and clay than theirneutral counterparts.

In aquatic environments, triclosan is expected to adsorb to suspended solids and sedimentsand may bioaccumulate (K ow 4.76), posing a concern for aquatic organisms.

Hydrolysis is not expected to be an important environmental fate process due to the

stability of triclosan in the presence of strong acids and bases. However, triclosan issusceptible to degradation via aqueous photolysis, with a half-life of <1 hour under abioticconditions, and up to 10 days in lake water. An atmospheric half-life of 8 hours has alsobeen estimated based on the reaction of triclosan with photochemically produced hydroxylradicals.

In the laboratory, triclosan degraded via aerobic soil metabolism and aerobic aquaticmetabolism, with half-lives of <4 days in soils and half-lives of <1.5 days (water layer) andup to 60 days (sediment and total system) in water-sediment systems."

Samsøe-Petersen et al . (2004) have described that half-life of triclosan for threeexperimental soils was calculated to be in the range of 17.4 to 35.2 days

Some observed concentrations of triclosan in the environment (e.g. Kumar et al . 2010) are

high enough to induce changes in the microbial population. However, the bioavailability oftriclosan in these environments (WWTP effluents, sludges, sediments, etc.) has not been



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determined. It is therefore important that the concentration effects of bioavailable triclosanare measured during the exposure period under study.

The presence of other chemicals (e.g. antibiotics, other biocides, surfactants…) in theenvironment may also affect the microbial population. Therefore it may be difficult to assess

the effect of triclosan alone against microbial populations in the environment. Triclosan-containing products are complex formulations since triclosan is poorly soluble inwater. The role of the formulations is important to ensure the bioavailability of triclosan.Formulations might also enhance biocidal activity and/or reduce microbial aggregation,improving the biocidal activity of the product. The bioavailability of triclosan in surfaces ortextiles, etc., is product dependent. Some manufacturers claim that triclosan does not leachout of their product.

8. MEASUREMENT OF RESISTANCE AND CROSS-RESISTANCE

Concentration is central to the definition of bacterial resistance in practice (McDonnell andRussell 1999, Maillard and Denyer 2009). Therefore, the measurement of bacterial lethalityrather than the measurement of bacterial growth inhibition is paramount. The determinationof the lethality of the in-use concentration of a biocide will indicate, by comparison to areference strain, whether a bacterial strain is insusceptible (i.e. intrinsically resistant) or hasacquired resistance to a biocide or not.

The determination of minimum bactericidal concentrations (MBCs) is also an appropriatemethodology that allows the comparison of lethality between a reference strain and

“resistant” clinical/environmental isolates. Here, the reference strains represent thepopulation of bacteria which is normally susceptible to the biocide. In addition thedetermination of the lethality of a biocide must involve the use of a neutralising agent or theremoval of the biocide. Failure to do so will provide an over-estimation of the lethality of thebiocide.

The determination of bacterial growth kinetics in the presence of a low concentration of abiocide can also provide indications to a change in bacterial phenotype (Thomas et al. 2004;Gomez Escalada et al. 2005a; Maillard 2007), but it does not indicate whether bacteria willbecome resistant to the biocide and cross-resistant to unrelated compounds or not.

Likewise, a number of protocols have been used to measure antibiotic susceptibility inbacterial isolates showing resistance, tolerance or increased insusceptibility to biocides orvice versa. The variety of protocols used contributes to the variability of the results reportedon antibiotic “resistance”. For example, some studies based a change in antibioticsusceptibility profile on measurement of zone of inhibition (Tattawasart et al. 1999; Thomaset al. 2005). More meaningfully studies used standardised antibiotic susceptibility

methodologies such as those given by the British Society for Antimicrobial Chemotherapy(BSAC) or Clinical and Laboratory Standards Institute (CLSI) to measure a change inantibiotic susceptibility profile. However a limited number of studies have looked at adecrease in antibiotic susceptibility that would be associated with treatment failure (Lear etal. 2006; Cottell et al . 2009).The effect of biocides on antibiotic susceptibility in bacteria hasbeen measured indirectly, whereby a bacterial population is treated first with a biocide andthe surviving bacteria then investigated for their susceptibility to antibiotics. However, thereare currently no well-referenced criteria or standard protocols for the evaluation of thecapability of a biocide to induce or select for resistance to antibiotics. Therefore, tools needto be developed to define for example the "minimal selecting concentration": the minimalconcentration of a biocide which is able to select or trigger the emergence/expression of aresistance mechanism that will confer clinical resistance to an antibiotic class in a defined

bacterium (SCENIHR 2009).

Since cross-resistance can be conferred by a number of distinct mechanisms, it is importantl h i f b i h h i Ad i



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modern genetic methods (e.g. PCR, -omics) and the development of an assay using specificchemosensitizers or markers (e.g. efflux pumps inhibitors) might allow the development ofroutine tests to identify resistance mechanisms.

9. DATA GAPS ON SCIENTIFIC KNOWLEDGE

In the course of this work, several important gaps were noted. These can be divided intoscientific and technical gaps:

9.1. Scientific gaps:

1. Environmental studies focussing on the identification and characterisation ofresistance and cross-resistance to antibiotics following use of triclosan.

2. In vitro studies to demonstrate whether triclosan, used at sub-lethal concentrations,

triggers the emergence of antibiotic resistance and/or select bacteria resistant toantibiotics. This has only been demonstrated in a limited number of bacterial genera.Further information for other genera should be obtained.

3. Despite in vitro evidence of the effect of triclosan on the emergence of antibioticresistance and on the selection of bacteria resistant to antibiotics, epidemiologicaldata indicating public health relevance are lacking.

4. There is no information available on the maintenance and transferability ofresistance and virulence markers in the presence of triclosan.

9.2. Technical gaps:

1. Standardisation of methodologies to measure resistance and cross-resistance isneeded.

2. Information on production, use volumes is required to assess the exposure ofbacteria to triclosan in various matrices.

3. Data on the fate and bioavailability of triclosan in the environment are sparse.Information on environmental concentrations, contact time, microbial populationpresent in the field and bacterial exposure, is insufficient to determine whetherexpression of resistance actually occurs in situ.

4. No validated methodologies are available for the determination of the dose-responserelationships and of the threshold triggering the emergence of antibiotic/biocide

resistance and/or the selection of resistant bacteria.

5. The role of bacterial biofilm in resistance to triclosan has been shown. Furthermore,bacterial biofilms are very common in the environment. Yet, most laboratories arenot using biofilm tests to assess the efficacy of biocides (Cookson 2005). There are,currently, no European standards for the testing of disinfectants against biofilms forhealth care applications.

A more detailed research strategy for investigating the antimicrobial resistance effect ofbiocides is presented in a separate opinion from the SCENIHR (2010).

10. RISK ASSESSMENT



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Triclosan is the most studied biocide with respect to antimicrobial resistance. Such a level ofinformation, notably on its activity on bacteria, the identification of mechanisms of microbialresistance, including genomic and proteomic aspects, is commendable. However, in spite ofthis level of information on mechanisms, information on the interaction between triclosanand microbial cells/communities including data on exposure and bioavailability in situ is

lacking. Thus, a full risk assessment of triclosan cannot be performed. However, a numberof points can be made:

- a hazard has been identified concerning the effect of triclosan on the regulation ofresistance genes in bacteria

- mechanisms which can promote resistance and cross-resistance to biocides andantibiotics in bacteria have been identified

- high concentrations of triclosan (compared to concentrations known to select forresistance in in vitro experiments) have been measured in certain environmentalcompartments, however a link with cosmetic or other specific product uses could not bemade.

- bacterial biofilms are widespread in the environment and are able to survive exposure toadverse environmental factors.

10.1. Limitation in activity

Bacteria can be classified according to their intrinsic resistance to biocides. Bacterialendospores are considered to be most resistant, followed by mycobacteria, Gram-negativebacteria and Gram-positive bacteria (Maillard 2005a). Triclosan is not sporicidal. It is notbactericidal against certain bacteria such as P. aeruginosa and Burkholderia sp. (Rose et al .2009).It might also have limited activity against certain mycobacteria as these micro-organisms are considered to be less susceptible to biocides than Gram-negative bacteria.

10.2. Genetic and bacterial point of view

Recent laboratory studies indicate that, during short exposures of mid-logarithmic growthphase to MIC concentrations (30 min at 0.12 mg/L), triclosan can trigger a genetic responsein Gram-negative bacteria (e.g. E. coli, S. enterica) inducing expression of genes involved inbiocide and antibiotic resistance. In addition, in Listeria monocytogenes triclosanconcentrations of 19 mg/L to 150 mg/L activate the expression of virulence factors(Kastbjerg et al . 2010).

Concerning the genetic aspects; genetic mobile elements play an important role in bacterialresistance response since they contain resistance genes (coding for pump, enzyme, qnrfactors, etc) which can confer resistance to different drug families. The gene pool encodingfor various mechanisms that confer resistance to antimicrobials has been shown to be

present in soil bacteria (Dantas et al . 2008). Although exposure to some biocides (such asquaternary ammonium compounds) favours the dissemination and maintenance of suchgenetic mobile elements in bacteria and subsequently may facilitate the exchange of keygenes between bacterial species (Paulsen et al . 1998, Pearce et al . 1999, Sidhu et al . 20012002, Bjorland et al . 2001, Noguchi et al . 2002), this has not been reported for triclosan.

10.3. Environment point of view

Several recent studies have clearly demonstrated the widespread presence of triclosan inthe environment, especially in wastewater, in wastewater treatment plant effluents, inrivers and in sediments. However, there is limited information from the EU. The reportedconcentrations range from less than 0.001 ng/L (seawater) to 133 mg/kg (biosolids fromWWTP) (see Table 4). The following information is also necessary for the risk assessment:

a) The bioavailability of triclosan in these environments,



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b) The microflora in contact with triclosan in these environmental compartments,

c) Whether this microflora contains bacterial species in which triclosan is able to trigger agenetic response. If not, could the environmental bacteria in contact with triclosan transfergenetic elements (containing resistance genes) to "target" bacteria?

Regulation cascades and corresponding resistance genes are present in the soil bacteria.These bacteria may both serve as original source/reservoir of genetic mobile elements(horizontal transfer) and as genetic manipulator (exchange between chromosomal andmobile genes) of resistance genes in the presence of a selective pressure.

10.4. Biofilm formation in specific environmental conditions

Bacterial biofilms are widespread in the environment including waters, plants, etc. Theydeserve a special attention because of three main characteristics: the decrease inbioavailability of antibacterial agents within the biofilm, the presence of dormant/persisterbacteria, and in complex biofilms the presence of various bacterial species in close contact

that facilitate exchange of genetic material.

11. CONCLUSIONS

Triclosan is the most studied biocide with respect to bacterial resistance. Such a level ofinformation, notably on its activity against bacteria, the identification of mechanisms ofmicrobial resistance including genomic and proteomic aspects, is commendable and shouldbe extended to other biocides. This information allows better understanding of triclosaninteractions with bacterial cells and should be applied to ensure that its use is sustainablefor human health. Based on the available scientific information, it is not possible to quantify

the risk of development of antimicrobial resistance induced by triclosan applications,including its use in cosmetics. However, there are environmental concentrations in anumber of geographically distinct areas high enough to suggest that triggering of bacterialresistance could also occur in the environment. The applications of triclosan whichcontribute to those high environmental concentrations cannot be properly identified norquantified at present. This should be taken into account when considering the current andfuture uses of triclosan in all applications so as to ensure that the demonstrable benefits forhuman health in certain applications are not compromised.

Low concentrations of triclosan can trigger the expression of resistance and cross-resistancemechanisms in bacteria in vitro. Some environmental concentrations reported in a numberof geographically distinct areas are high enough to give plausibility to this scenariooccurring outside of the laboratory and warrant further investigation. The presence of other

chemicals (e.g. antibiotics, surfactants, other biocides, etc.) in the environment, which mayalso affect microbial populations, would preclude assessing the effects of triclosan alone.

The emergence of resistance induced/selected by triclosan is related to the genetic controlon the resistance gene(s) present on chromosomal and genetic mobile elements in vitro.This represents the origin for a hazard about selection and dissemination of cross-resistancewith other anti-bacterial molecules including biocides and antibiotics.

Bacterial biofilms are widespread in the environment including waters, plants, etc. Theydeserve special attention because of three main characteristics: the decrease inbioavailability of antibacterial agents within the biofilm, the presence of dormant/persisterbacteria, and in complex biofilms the presence of various bacterial species in close contactthat facilitates some genetic exchange.

Triclosan, like any other biocide, contributes to the selection of less susceptible bacteria in acomplex microcosm in vitro. The impact of such a selection is unclear, as is the fitness ofth “ l t d” b t i l i f ll i t i l Th f i it t di



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investigating long-term triclosan exposure (i.e. at least 6 months) did not indicate changesin resistance susceptibility in the predominant bacteria selected for monitoring but thechanges in the entire flora were not evaluated.

There is so far no epidemiological data linking outbreaks of antimicrobial resistant human

and zoonotic pathogens following exposure to triclosan from cosmetics and other products.When used appropriately, biocides, including triclosan, have an important role to play indisinfection, antisepsis and preservation. Information on the expression/triggering ofbacterial resistance mechanisms should be considered to (re-)assess the uses of triclosan inorder to preserve its efficacy.

Where biocides, including triclosan, are used intensely, monitoring for emerging resistancein the microbial flora should be conducted.

12. OPINION

Does the SCCS consider a continued use of triclosan as a preservative in cosmetic products

as safe taking into account the new provided documentation of resistance development bycertain micro-organisms and cross-resistance?

At present, several distinct hazards have been identified: (i) the effect of triclosan on thetriggering/regulation of resistance genes in bacteria (ii) the existence of mechanisms whichcan promote resistance and cross-resistance to biocides and antibiotics in bacteria, (iii) highconcentrations of triclosan (compared to concentrations known to select for resistance in invitro experiments) have been measured in certain environmental compartments and (iv)bacterial biofilms are widespread in the environment and are able to survive exposure toadverse environmental factors. The first two of these hazards have been identified in vitro.

The presence of resistance genes in soil bacteria should be investigated further.

The six in situ studies and the one meta-analysis quoted in this document have failed todemonstrate an increase in antibiotic resistance following triclosan use. While these resultsare at first sight reassuring, the differences of methodologies used to measure “resistance”and to analyse the data make it premature at this stage to conclude that triclosan exposurenever leads to developing microbial resistance. These studies were state-of-the art at thetime they were performed but they did not have the modern tools (e.g. proteomic orgenomic analysis) available today to investigate the complete bacterial population and thebacterial response to biocides. These useful in situ studies do not provide information onexpression of genes involved in resistance, maintenance of resistance and virulence genesand transfer of resistance determinants. Thus the SCCS strongly recommends performingadditional in situ studies looking at these aspects and bacterial phenotypes where known

concentrations of triclosan have been found in the environment.

This opinion concerns the safety of triclosan in terms of microbiology, i.e. generation ofbacterial resistance harmful for human health. Based on the available scientific informationincluding recent data from in vitro investigations (proteomic and genomic analyses), it is notpossible to quantify the risk associated with triclosan (including its use in cosmetics) interms of development of antimicrobial resistance (i.e. selection for less susceptiblepopulation), genetic basis for resistance and dessemination of resistance. In view of theconcentrations of triclosan reported to trigger resistance in vitro, some of the environmentalconcentrations found in a number of geographical distinct areas are high enough to suggestthat bacterial resistance could be triggered. However, no studies have been conducted onthis aspect. The applications of triclosan which contribute to those high environmental

concentrations cannot be properly identified nor quantified at present and the presence ofother chemicals (e.g. antibiotics, surfactants, other biocides, etc.) in the environment,which may also affect microbial populations, would preclude assessing the effects of

i l i d d l



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Due to the limited number of in situ studies of resistance induced by triclosan to date, SCCScan only recommend the prudent use of triclosan, for example in applications where ahealth benefit can be demonstrated. However, conclusions from in vitro studies cannot beignored, notably the role of triclosan (and other biocides) in triggering resistance and in thedissemination (or lack of) resistance determinants. Hence, the SCCS appreciates that

research investment from industry will be maintained to contribute to a betterunderstanding of the potential risks associated with triclosan applications. Research intriggering mechanisms of resistance, maintenance of the gene pool and the transfer ofresistance and virulence determinants, and improving the translational application oflaboratory results to situations in situ are needed.

13. COMMENTS RECEIVED DURING THE PUBLIC CONSULTATION

A public consultation on this opinion was opened on the website of the EU non-food

scientific committees from 29 March to 26 May 2010. Information about the publicconsultation was broadly communicated to national authorities, international organisationsand other stakeholders.

In total, 10 contributions were received of which 5 were from public authorities, 3 fromindustry and two from individuals with professional links to this issue.

Each submission to the public consultation was carefully considered by the Working Groupand responses were formulated for each.The opinion has been revised to take account of allthe relevant comments and the literature has been updated with relevant publications. Thescientific rationale and the opinion were clarified and strengthened in certain respects. Theoverall opinion, however, remains unchanged.

14. MINORITY OPINION

None



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Personal Care Products CouncilCommitted to Safety,

Quality Innovation

Memorandum

TO: F. Alan Andersen, Ph.D.

Director COSMETIC INGREDIENT REVIEW CIR

FROM: Triclosan Review Subcommittee of the CJR Science and Support Committee

DATE: Mayll,2010

SUBJECT: Comments on the Scientific Literature Review on Triclosan

The Triclosan Review Subcommittee of the CR Science and Support Committee is concerned that this

draft of the Triclosan report is still incomplete. The attached comments should be addressed

before the CIR Expert Panel is provided a draft of the Triclosan report to review. Therefore,

please delay the review of this report until at least the August 30-3 1, 2010 CR Expert Panel

meeting.

References that still need to be added to the report:

2009 SCCP Opinion on Triclosan available from the On-Line

Ailmyr M, Panagiotidis G, Sparve E, et al. 2009. Human exposure to triclosan via toothpaste

does not chinge CYP3A4 activity or plasma concentrations of thyroid hormones. Basic

Clin Pharmacol Toxicol. 105 5 :339-44.

Bagley D, Lin Y. 2000. Clinical evidence for the lack of triclosan accumulation from daily use

in dentifrices. Am J Dent 13: 148-152. attached

Calafat A, Ye X, Wong L, Reidy J, Needham L. 2008. Urinary concentrations of triclosan in

the U.S. population: 2003-2004. Envion Health Perspect 116: 303-307.

Crofton, K; Paul K, DeVito M, Hedge J. 2007. Short-term in vivo exposure to the water

contaminant triclosan: Evidence for disruption of thyroxine. Env. Tox. Pharm 24 :

194-197.

Dayan A. 2007. Risk assessment of triclosan [Irgasan] in human breast milk. Food Chem

Toxicol 45 : 125-128. attached

Rodricks JV, Swenberg JA, Borzelleca JF, et al. 2010. Triclosan: A critical review of the

experimental data and development of margins of safety for consumer products.

Critical Reviews in Toxicology 40 5 : 422-484.

More information on exposure to Triclosan can be found in the 2009 SCCP opinion on Triclosan, theRodricks et al. 2010 review, and the CDC biomonitoring study Calafat et al., 2008 .

The expectation is that the CR Expert Panel will limit Triclosan use in cosmetics to USP grade

material.

The Rodricks et al. 2010 review provides an overview of the Triclosan carcinogenicity data.

Endocrine activity of Triclosan is reviewed in the following unpublished review attached .

1101 17th Street, N.W., Suite 3OO Washington, D.C. 20036-4702 202.331.1770 202.331.1969 fax www.personalcarecouncil.org




p.32-33 Please provide more details of these studies e.g. the tu l duration of the study numbers of

animals more details about the o ra l route of exposure gavage diet drinking water .

p.36 Reference 35 Please correct “thyrozine” to”thyroxine”?

4




Personal Care Products CouncilCommitted to Safety,

Quality Innovation

Memorandum

TO: F. Alan Andersen, Ph.D.

Director COSMETIC ThGREDIENT REVIEW CIR

FROM: John Bailey, Ph.D. J__ 9.i bIndustry Liaison to the CW Expert Panel

DATE: May 17, 2010

SUBJECT: Comments on the Scientific Literature Review on Triclosan

Enviorn International Corporation. 2 1 Comments on the Scientific Literature Review Safety of

Triclosan as a Preservative in Cosmetics.

1101 17th Street, N.W., Suite 300 Washington, D.C. 20036-4702 202 .331 .1770 202 .331 .1969 fax www.personalcarecouncil.org




Comments on the Scientific Literature Review

Safety of Triclosan as a Preventative in Cosmetics

Dated: April 9, 2010

Prepared by:

ENVIRON International Corporation

May 11 , 2010

General Comment:

The Cosmetic Ingredient Review group ha s released entitled, “Scientific LiteratureReview on the Safety of Triclosan as a Preventative in Cosmetics”. As noted, preparation

of this Scientific Literature Review SLR document relied on reviews prepared by

various government agencies, especially that prepared by the Australian Government

Department of Health and Ageing NICNAS , the United States Environmental

Protection Agency USEPA and US National Toxicology Program NTP . A more

recent, comprehensive review of the triclosan toxicity and pharmacokinetic data which

relied on a review of the prime literature, should be considered Rodricks et al. 2010 .

While the SLR clearly states that the CW ha s relied extensively on reviews already

available from various governmental sources, some review of primary literature is needed

to insure that study results have been summarized correctly.

Specific Comments:

Comments on CIR Toxicokinetic section Section IV

Both in vivo and in vitro studies have been conducted in human volunteers and in

animals to evaluate the potential dermal absorption of triclosan following single or

repeated applications of various products and formulations, as reviewed by Rodricks et

al. 2010 . In vitro dermal absorption studies with human sk in to which several triclosan

containing products, e.g. body lotion, were used to provide estimates of the amount of

triclosan delivered from dermal application Rodricks et al. 2010 . These results were

compared to triclosan levels reported in the latest NHANEs study. In this SLR relianceon this single study to derive an estimate of systemic exposure, expressed in igIkgJday, is

likely to result in an overestimate of that exposure.

• p. 11, Absorption/Toxicokinetics While a single plasma level as high as 229

ng/ml was reported in one volunteer, this level was the maximum concentration

measured in one out of 13 volunteers on day 20 of a pilot study designed to

evaluate dermal absorption of triclosan from a specific use pattern of a handwash




product Ciba Specialty Chemicals Corporation 2002). The mean plasma

concentration for the entire group of volunteers was approximately 73 ng/ml

mean value included the one outlier value of 229 ng/ml ; the minimum

concentrations were betow the limit of quantitation.

• 1, Absorption/Toxicokinetics The estimate of plasma levels associated withleave-on cosmetics 1.15 to 3.82 igIml appears to be based on a factor of 3 to 10

less than the estimated plasma concentration from whole body exposure to a

handwash 11.45 pg/mI , rather than 3 to 10 less than seen in handwashes <229

ng/ml . This clearly is an overestimate of the amount of triclosan levels in plasma

resulting from leave-on cosmetics, which would be expected to cover only a

fraction of the body. In addition, these triclosan plasma levels would be expected

to be similar to that measured from contact with a handwash product. This is

critical if these plasma levels were to be compared with plasma levels from

toxicity studies in an attempt to evaluate the potential for health effects from

leave-on cosmetics.

• Distribution The available information from rodent radioactivity studies

suggests similar distribution patterns in the hamster and the rat, with no evidence

of accumulation in tissues. In contrast to this, distribution of triclosan in the

mouse is different, with evidence of accumulation in the liver observed Rodricks

et al. 2010).

• P.11, Metabolism For most species, including humans, the glucuronide

metabolite is the predominant metabolite at lower concentrations; however, in the

case of the mouse and the dog, the sulfate conjugate is the dominant metabolite

formed. The metabolism shift from the generation of predominantly glucuronide

conjugates to sulfate conjugates depending upon triclosan concentrations is notobserved in the mouse van Dijk 1995; Rodricks et al. 2010).

Comments on the Reproductive and Developmental Toxicity Section Section V):

On p. 12, the document states that triclosan’s potential reproductive/developmental

effects “indicate that triclosan is not highly toxic for these endpoints”. This statement is

misleading and may imply to the reader that triclosan has reproductive and

developmental effects at some high dose. The reproductive and developmental studies

conducted with triclosan do not support such a conclusion.

• On p. 13, under “Reproductive Toxicity”, the SLR states that in the two

generation rat study triclosan produced both “reproductive and systemic

effects” at the highest dose tested, 150 mg/kg/day. That statement is

inaccurate. This study was reviewed in detail in Rodricks et al. 2010).




o No effects on reproductive function were reported in either the FO orFl parental generations nor were consistent or dose-related indices of

pup health and survival in the Fl and F2 offspring.

o Decreases in pup body weight in offspring of dams receiving the

highest dose of triclosan 150 mg/kg/day were noted in the Flgeneration pups after Post-natal day 14 but returned to control levels at

later time point. Pup body weights were not affected at in any group

in the F2 generation. Transient changes in body weight not

accompanied by any changes in reproductive competence should not

be classified as a “systemic” effect.

• On p. 13 under “Developmental Toxicity”, the SLR cites the USEPA review

for information on developmental endpoints.

o No discussion of developmental toxicity studies is provided in the

EPA 2008 document cited in the SLR. The EPA 2008 documentcites a separate EPA document entitled “5-Chloro2- 2,4-

dichlorophenoxy phenol triclosan : Toxicology Chapterfor the

Reregistration Eligibility Decision RED document” and dated August29, 2008 as providing this information. However, this document couldnot be readily located on the EPA website www.epa.gov .

o The basis for the NOAEL of 50 mg/kg/day cited by NICNAS 2009 is

unclear. CIR incorrectly suggests that it is based on a mouse study

that showed maternal toxicity, bu t not developmental toxicity;

however, this is also incorrect. The study discussed is a mouse

developmental study conducted at Argus Research Laboratories 1992in which pregnant CD-i mice were given triclosan in the diet ongestation days GD 6 through 15 at concentrations resulting in doses

of 0, 10, 25 , 75, or 350 mg/kg/day; therefore this study does notprovide the basis for a maternal NOAEL of 50 mg/kg/day.

Comments on the Endocrine Disruption Section Section V

On pgs.14 and 15, the SLR has a section on the potential endocrine disruption action of

triclosan. The section relied heavily on two in vitro studies and did not integrate the fulltoxicity data based, especially the data generated in chronic bioassays in mice, rats, and

hamsters with the results from short-term studies of changes in thyroid hormone levels.

• The SLR relied on the in vitro studies conducted by Gee et al. 2008 and Ahn etal. 2008 that investigated triclosan interaction with estrogenic and androgenic

receptors; however, a number of issues must be considered.

o While these in vitro studies suggest potential activity of triclosan at certain

receptor sites, it must be considered that the cells used in some of these

experiments were conducted in immortalized or cancer cell lines that are




not expected to respond in a similar manner to normal or primary cells

Gentry et a . 2010).

o aclclition cc I lines, such as those drrvcc1 from the kidney, are not the

target organ of concern and may not behave in a manner similar to cells

from the appropriate organ.

o The concentrations of tnclosan and endogenous steroids used in some of

these assays i.e., Chen et al. 2007 were selected by the authors to achieve

a maximum response and are not similar to those expected in v ivo.

o It should be noted that no effects on reproduction or development were

reported in the battery of tests for tnclosan conducted in vivo in rats at

doses of tnclosan ranging from 15 to 150 mg/kg in a two-generation

reproductive study Morseth 1988) or at doses of 15 to 300 mg/kg/day in

one-generation developmental studies Schroeder and Daly 1992a;

Denning et al. 1992). In addition, no developmental effects have beennoted in mice administered doses up to 25 mg/kg/day Christian and

Hoberman 1992), or in rabbits administered doses up to 50 mg/kg/day

Schroeder and Daly 1992b) or in hamsters administered doses up to 80

mg/kg/day Piekacz 1978).

o Therefore, these in vitro results must be interpreted with caution when

extending them to the whole animal.

• The SLR also discusses the potential effects of triclosan on thyroid hormone

levels. Again a number of issues must be considered before drawing conclusions

as to the relevance of these short-term studies reporting changes in thyroidhormone levels in the absence of evidence of toxicity.

With regard to the potential effects on the thyroid:

• The short-term studies conducted in rats report decreases in one of the three key

thyroid hormone levels T4) when triclosan was administered at very high doses

up to 300 to 1000 mg triclosan/kg body weight/day Crofton et al. 2007; Paul et

al. 2009 2010; Zorrilla et al. 2009 .

• Levels of TSH were not affected in any of these studies, indicating a lack of direct

effect on centrally-mediated control of thyroid function but rather likely to be theresult of altered levels of liver enzymes.

• No signs of altered thyroid function was seen in any of the toxicity studies

conducted in several species for durations up to two years:

o No overt signs of thyroid-related toxicity in experimental animals or

human volunteers,

4




o No signs of continued pressure on thyroid homeostasis, such as the

production of thyroid hyperplasia or neoplasia, and

o No signs of altered thyroid homeostasis during critical windows of

development from in utero exposure.

• It should be noted that changes in thyroid hormone levels in rats do not

necessarily mean that thyroid function in rats will be altered or that physiological

systems in rats will be adversely affected. Nor do these changes in serum thyroid

hormone levels in rats translate quantitatively to similar changes in humans

because of the differences between rats and humans in both the capacity to keep

thyroid hormones within normal physiological ranges but also because of thedecreased sensitivity of humans to these changes.

• Further, no changes in thyroid hormone levels were measured in human

volunteers who brushed twice daily with a triclosan-containing toothpaste triclosan concentration was 0.3 w/w for 14 days resulting in an estimated

intake typical for the use of triclosan-containing toothpaste of 0.01 mg/kg/day .

With regard to the potential from reproductive/developmental effects:

• As noted above for the changes in serum thyroid hormone concentrations,

changes in levels of circulating hormones associated with male or female

reproductive systems in rats serve as a screen for the potential of a xenobiotic to

have an adverse impact on reproductive/developmental competency in humans.

Such changes in these screening assays should be viewed in parallel with in vivo

tests for reproductive/developmental effects, when conducted as they have fortriclosan, to determine if changes in hormone levels measured in these screening

studies were sufficient to affect function.

• Two studies were conducted in rats with considerably different results reported

Kumar et a . 2009; Zorrilla et al. 2009 . Both Kumar et al. (2009) and Zomlia et

al. 2009 evaluated changes in LH and testosterone concentrations, both

indicators of a potential impact on steroidogenesis.

o Kumar et al. 2009 reported significant decreases in LH, testosterone, and

testes weight, while Zorrilla et al. 2009 did not.

o Zorrilla et al. (2009) did not see any histopathological changes in thereproductive tissues examined. More importantly, Zomlla et al. 2009

reported no change in the age of onset of preputial separation, an indicator

of the onset of puberty, which would have been expected if the changes in

serum hormones, i.e., testosterone were impacted.

5




o Several methodological flaws in the Kumar et al. 2009 raise questions

about these results and their application to humans

• While Kumr ct al. 2009 measured channes in endocrine levels associated ‘cith

normal functioning of the reproductive/developmental system, no evidence of an

impact on this organ system in animals has been demonstrated after chronicexposure in rats Yau and Green 1986 , in a two-generation reproductive study in

rats Morseth 1988 , or in developmental studies in rats Schroeder and Daly

1992a; Denning et al. 1992 or in mice Christian and Hoberman 1992 , hamsters

Piekacz 1978 or rabbits Schroeder and Daly 1992b .

• The lack of reproductive/developmental effects reported in several tnclosan

studies in experimental animals do not support the potential for triclosan to result

in adverse reproductive effects in rats or humans. More importantly, humans have

high levels of naturally occurring estrogen and testosterone. swith thyroid

hormones, humans have a critical buffering capacity to bind these hormones and

ac t as a reserve to release free biologically active estrogen or testosterone, as wellas critical feedback loops involving the pituitary-hypothalamic axis to respond to

decreased or increased levels of estrogen or testosterone. Both the capacity in

humans to bind and release these hormones, with a much higher capacity in

humans than in rodents, and the feedback loops act to maintain homeostasis. As

with the thyroid, changes in hormone status that could occur with exposure to a

xenobiotic can be compensated for in humans by these active systems that

maintain homeostasis, especially at environmentally relevant exposures to

triclosan.

Comments on the Carcinogenicity Section Section V

On p. 16 the SLR correctly stated that triclosan is not considered “likely to be a human

carcinogen”.

• On p. 16, first paragraph, the SLR states that the FDA considered the datainadequate to address the question of carcinogenicity by the oral route ofexposure. It is clear, however, that tnclosan was not carcinogenic when

administered to r ats and hamsters in the diet resulting in internal doses that would

exceed by possibly orders of magnitude that received by the dermal route from

the use of tnclosan-containing products.

• The SLR correctly reports that USEPA has concluded that triclosan whenadministered in the rat and hamster 2-year dietary studies was not carcinogenic.

Further the USEPA concluded that increase in the incidence of liver cancer in the

mouse 2-year dietary study occurred by a PPARCL mode of action and was “not

likely to be carcinogenic in humans”.

6




2006; McBain et al. 2 4; Sullivan et al. 2003; and Walker et aT 1994 ; and one

meta-analysis Aiello et al. 2004 have failed to demonstrate an increase in

antibiotic resistance following triclosan use. Although drawing similar

conchisions to the seven studies i l ntifi r hove. the CIR SLR only rites the

studies by Aioello et aT 2004 , Cole et al. 2003 and Walker et aT 1994 , the

CIR SLR should review and cite the additional in situ studies as there is a limitednumber available.

• There is a reference to”2009 SCENIHR”, further in the summary a reference to”

a more recent opinion by SCENIIIR”, both refer to the same reference. The

second reference is probably the 2 Draft Preliminary Opinion. The reference

should be checked and revised accordingly.




References

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and susccp lit les or bacteria isoatcd rrum hands in the community. ‘nit; cmhial

Agents chemotherapy. 48: 2973-2979.

Aiello, A. E., E. L. Larson, and S. B. Levy. 2007. Consumer antibacterial soaps:

Effective or just risky? Clinical Infectious Diseases. 45 :S 137-47.

Bailey, A.M. C. Constantinidou, A. Ivens, M.I. Garvey, M.A. Webber, N. Coidham,

J.L. Hobman, I. Wain, M.J. Woodward, and L.J.V. Piddock. 2009. Exposure of

Escherichia coli and Salmonella enterica serovarTyphimurium to triclosan induces a

species-specific response, including drug detoxification. Journal of Antimicrobial

Chemotherapy. 64 , 973-985.

Chen, J., Ahn, K., Gee, N., Gee S., Hammock, B., and B. Lasley. 2007.

Antiandrogenic properties of parabens and other phenolic containing small moleculesin personal care products. Toxicology and Applied Pharmacology 22 1 3 278-284.

Christian, M., and Hoberman, A. 1992. Developmental Toxicity Embryo-Fetal

Toxicity and Teratogenic Potential Study of C-P Sample No. 38326 Administered

Orally Via the Diet to Crl:CD-1 ICR BR Presumed Pregnant Mice. Argus Research

Laboratories,. Protocol number 403-0 10. Sponsors Study number 92-001.

Ciba Speciality Chemicals Corporation. 2002. A pilot study for the in vivo

evaluation of the percutaneous absorption of triclosan. Unpublished report No: CIBA

03-01-013 Arizona Clinical Research Centre, Tuscon, Arizona, USA.

Cole E. C., R. M. Addison, J. R., Rubino, K. E. Leese, P. D. Dulaney, M. S. Newell,

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